Method of nanopore sequencing of concatenated nucleic acids

ABSTRACT

The invention relates to a new method of characterising two or more target polynucleotides using a pore. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of international application number PCT/GB2017/051493, filed May 25, 2017, and claims the benefit of GB application number 1609221.5, filed May 25, 2016, each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a new method of characterising two or more target polynucleotides using a pore.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.

When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In the strand sequencing method, a single polynucleotide strand is passed through the pore and the identities of the nucleotides are derived. Strand sequencing can involve the use of a molecular brake to control the movement of the polynucleotide through the pore.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that it is possible to improve the efficiency of transmembrane pore-based characterisation of two or more target polynucleotides by sequentially attaching the target polynucleotides together to form a concatenated polynucleotide. If the binding of a second target polynucleotide to a first polynucleotide occurs selectively in the sense that binding only occurs as the first target polynucleotide moves through the pore, the time taken for the second target polynucleotide to contact the pore is reduced and binding of the target polynucleotides present in a sample in the absence of the pore is avoided. Concatenation of the target polynucleotides is only achieved when a first target polynucleotide moves through a pore. This is important because concatenation of the target polynucleotides in solution would result in a reduction of the number of free ends that could be captured by a pore.

Accordingly, the invention provides a method of characterising two or more target polynucleotides. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide. The method comprises:

(a) contacting a first target polynucleotide with a transmembrane pore in a membrane such that the first target polynucleotide moves through the pore;

(b) sequentially attaching to the first target polynucleotide one or more subsequent target polynucleotides to provide a concatenated polynucleotide within which the target polynucleotides move through the pore in attachment order, wherein a subsequent target polynucleotide is selectively attached to the preceding target polynucleotide in the attachment order when the preceding target polynucleotide moves through the pore; and

(c) taking one or more measurements which are indicative of one or more characteristics of the concatenated polynucleotide as it moves with respect to the pore.

Also provided is a population of two or more polynucleotide Y adaptors for characterising two or more double stranded target polynucleotides, wherein each adaptor comprises first and second parts which are capable of hybridising together and wherein each first part is initially protected from hybridisation to the second part.

In addition, a kit for characterising two or more double stranded target polynucleotides comprising a population of Y adaptors of the invention and a population of hairpin loops is provided.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the attachment of a subsequent target polynucleotide (labelled B) to the first target polynucleotide (labelled A) to produce a concatenated polynucleotide. The attachment point in the first target polynucleotide was revealed for attachment as the first polynucleotide moved through the pore (labelled C).

FIG. 2 shows example current traces (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where (ΔM1)G1G2=deletion of M1 and then addition G1 and G2)) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The top trace shows the controlled translocation of the concatenated polynucleotide and the lower trace labelled 1 shows zoomed in region 1 of the top trace.

FIG. 3 shows example current traces (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The top trace shows the controlled translocation of the concatenated polynucleotide and the lower trace labelled 2 shows zoomed in region 2 of the top trace.

FIG. 4 shows example current traces (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The top trace shows the controlled translocation of the concatenated polynucleotide and the lower trace labelled 3 shows zoomed in region 3 of the top trace.

FIG. 5 shows example current traces (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The top trace shows the controlled translocation of the concatenated polynucleotide and the lower trace labelled 4 shows zoomed in region 4 of the top trace.

FIG. 6 shows example current traces (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The top trace shows the controlled translocation of the concatenated polynucleotide and the lower trace labelled 5 shows zoomed in region 5 of the top trace.

FIG. 7 shows the attachment of a subsequent target polynucleotide (labelled B) to the first target polynucleotide (labelled A) using click chemistry (see click chemistry linkage labelled D) to produce a concatenated polynucleotide. The attachment point in the first target polynucleotide was revealed for attachment as the first polynucleotide moved through the pore (labelled C).

FIG. 8 shows an example current trace (y-axis label=Current (pA), x-axis label=Time (s) for both traces) of when a helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of the concatenated polynucleotide through an MspA nanopore. The first polynucleotide had 5 subsequent target polynucleotides attached to it using click chemistry. The regions of the current trace which correspond to either the template or complement region of the first polynucleotide (T1 and C1) and each subsequent polynucleotide (T2-6, C2-6) as they translocated through the nanopore are identified. Spacers in the leader and the hairpins of the polynucleotides allow more current to flow and produce a spike in current as they translocated through the nanopore. The spacers are highlighted with *(a-k) and mark transitions between the template and complement regions of the first and subsequent polynucleotides which were attached to form the target concatenated polynucleotide.

FIG. 9 shows the attachment of a subsequent target polynucleotide (labelled B) to the first target polynucleotide (labelled A) to produce a concatenated polynucleotide. The attachment point in the first target polynucleotide was revealed for attachment as the first polynucleotide moved through the pore (labelled C). In this example the first polynucleotide has one type of click chemistry reactive group (for example azide reactive groups) at either end and the subsequent polynucleotide has a different type of click chemistry reactive group (for example DBCO reactive groups) at either end. The enzyme used to control the movement of the DNA through the nanopore is not shown on this figure.

FIG. 10 shows how only template strands of target polynucleotides can be characterised and concatenated, where the method of attachment used to join the polynucleotides together is click chemistry. The concatenation adapter complex contains a motor protein and a release protein. This adapter ligated to both ends of a target polynucleotide. Both proteins are stalled on the ligated adapter complex until the adaptor ligated to a target polynucleotide is captured by the pore. Once a first polynucleotide has been captured, the blocking chemistry used to stall the proteins is overcome by both proteins. The motor protein then controls the interaction of the first polynucleotide with the pore, as described previously, and the release protein, which can translocate more quickly than the motor protein, separates the strands to expose a sequence (3′ hybridisation site) in the 3′ end of the adaptor linked to the end of the target polynucleotide that is complementary to a 5′ nucleic acid sequence (5′ hybridisation site) of the leader strand of an adapter complex that is ligated to a second target polynucleotide. With the 3′ hybridisation site revealed, the 5′ hybridisation site in the second target polynucleotide can then hybridise to the revealed 3′ hybridisation site and covalent coupling of the 3′ end of the first polynucleotide to the 5′ of a the second polynucleotide can occur. This process then repeats for further concatenation of target polynucleotides.

It is to be understood that Figures are for the purpose of illustrating particular embodiments of the invention only, and are not intended to be limiting.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer of α-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19): 7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNA polymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derived from the sbcB gene from E. coli. It encodes the exonuclease I enzyme (EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derived from the xthA gene from E. coli. It encodes the exonuclease III enzyme from E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease III enzyme from E. coli. This enzyme performs distributive digestion of 5′ monophosphate nucleosides from one strand of double stranded DNA (dsDNA) in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′ overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derived from the recJ gene from T. thermophiles. It encodes the RecJ enzyme from T. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T. thermophilus (TthRecJ-cd). This enzyme performs processive digestion of 5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzyme initiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derived from the bacteriophage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer. The enzyme performs highly processive digestion of nucleotides from one strand of dsDNA, in a 5′-3′ direction (www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on a strand preferentially requires a 5′ overhang of approximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of He1308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of He1308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of He1308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of He1308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows the codon optimised polynucleotide sequence encoding the wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence.

SEQ ID NO: 27 shows the amino acid sequence of the mature form of the wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence. The abbreviation used for this CsgG=CsgG-Eco.

SEQ ID NOs: 28 to 42 show the sequences used in the Examples.

It is to be understood that sequences are not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes two or more polynucleotides, reference to “an anchor” refers to two or more anchors, reference to “a helicase” includes two or more helicases, and reference to “a transmembrane pore” includes two or more pores and the like.

In this specification, where different amino acids at a specific position are separated by the symbol “/”, the / symbol “/” means “or”. For instance, P108R/K means P108R or P108K. In this specification where different positions or different substitutions are separated by the symbol “/”, the “/” symbol means “and”. For example, E94/P108 means E94 and P108 or E94D/P108K means E94D and P108K.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method of the Invention

The methods devised by the inventors have various advantages. In known methods of polynucleotide characterization using a transmembrane nanopore, the pore is open for some time after characterising one polynucleotide before a subsequent polynucleotide contacts the pore. The new method significantly reduces the open pore time between polynucleotides because the subsequent polynucleotide is being recruited as the preceding polynucleotide is being characterised. In some embodiments, open pore time may be abolished, substantially abolished or minimised. Not only does a reduced open pore time mean that the pore is doing more characterisation (processing more polynucleotides) than a pore in a conventional method, but it also means that there is a reduced chance of pore blocking (the likelihood of the pore becoming blocked is low, or relatively low compared to in conventional methods). Pore blocking tends to occur when there is an open pore state. If a polynucleotide strand is in the pore then there is a reduced chance of blocking occurring as the pore is already “occupied”. Therefore, if the open pore state time is reduced the chance of blocking is also reduced. A reduced open pore also means that the concentration of polynucleotides needed for characterisation is also reduced.

Any number of target polynucleotides can be investigated or characterised using the invention. For instance, the method of the invention may concern characterising two or more polynucleotides, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more 100,000 or more, 1,000,000 or more or 5,000,000 or more, polynucleotides. In particular, 2 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1,000,000 or more or 5,000,000 or more subsequent target polynucleotides are attached to the first target polynucleotide.

The two or more target polynucleotides may be different from one another. The two or more polynucleotides may be two or more instances of the same polynucleotide. This allows proof reading. The two or more target polynucleotides may be derived from the fragmentation of a longer target polynucleotide.

Concatenated Polynucleotide

The method comprises sequentially attaching to a first target polynucleotide one or more subsequent target polynucleotides to provide a concatenated polynucleotide. The concatenated polynucleotide comprises at least two target polynucleotides attached together.

The target polynucleotides move through the pore in attachment order. For instance, the first target polynucleotide moves through the pore immediately before the second target polynucleotide. The second target polynucleotide moves through the pore immediately before the third target polynucleotide and so on. In other words, the first target polynucleotide moves through the pore followed by the second target polynucleotide. The second target polynucleotide moves through the pore followed by the third target polynucleotide and so on.

Attachment

As target polynucleotides move through a pore, a subsequent target polynucleotide is selectively attached to the preceding target polynucleotide in the attachment order when the preceding target polynucleotide moves through the pore. For instance, the second target polynucleotide is selectively attached to the first target polynucleotide when the first target polynucleotide moves through the pore. The third target polynucleotide is selectively attached to the second target polynucleotide when the second target polynucleotide moves through the pore and so on.

In other words, the target polynucleotides are not attached to one another until a first target polynucleotide interacts with the pore. The interaction of a first target polynucleotide with the pore facilitates the attachment of the first target polynucleotide to a second target polynucleotide. The interaction with the pore may result in a conformational change in the first polynucleotide, such as for example dehybridisaton/separation of the two strands of a double stranded target polynucleotide, or removal of a protecting molecule to reveal or expose a site on the first polynucleotide that can interact with a site on the second polynucleotide. After the said site on the first polynucleotide is revealed or exposed, the first polynucleotide attaches to the second polynucleotide. The occurrence of the attachment only when the first polynucleotide interacts with the pore is referred to herein as “selective attachment”. This is distinct from “selective hybridisation” which is used herein to describe base pair binding between complementary regions of a first polynucleotide and a second polynucleotide.

Thus, the attachment is selective in the sense that a subsequent target polynucleotide cannot attach to the preceding target polynucleotide in the absence of the pore or until the preceding target polynucleotide moves through the pore. For instance, the attachment is selective in the sense that the second target polynucleotide cannot attach to the first target polynucleotide in the absence of the pore or until the first target polynucleotide moves through the pore. The third target polynucleotide cannot attach to the second target polynucleotide in the absence of the pore or until the second target polynucleotide moves through the pore and so on.

Selective attachment may be achieved in any way. A part of the preceding target polynucleotide (e.g. a site on a first polypeptide which binds to a site on a second polypeptide) is initially protected from attachment to the subsequent target polynucleotide and is revealed for attachment as the preceding target polynucleotide moves through the pore. For instance, a part of the first target polynucleotide is initially protected from attachment to the second target polynucleotide and is revealed for attachment as the first target polynucleotide moves through the pore. A part of the second target polynucleotide is initially protected from attachment to the third target polynucleotide and is revealed for attachment as the second target polynucleotide moves through the pore and so on.

The part may be protected in any way. The part is typically protected by a molecule which prevents attachment. Movement of the preceding target polynucleotide through the pore may remove the molecule and reveal the part for attachment. Any molecule may be used. For instance, a molecule may occlude one of the click reactive groups discussed below. This might be, for example, pyrene to stack with the DBCO. If the part comprises Ni-NTA groups (which can attach to polyhistidine, such as 6×His, in the subsequent target polynucleotide), the part may be protected with polyhistidine, such as 6×His, in the same target polynucleotide and vice versa, i.e. the part comprises polyhistidine, such as 6×His, and is protected by Ni-NTA groups. If the part comprises cyclodextrin (which can attach to amantadine in the subsequent polynucleotide), the part may be protected by amantadine in the same target polynucleotide or vice versa. The part and the protecting molecule may be present on opposite strands of a double stranded target polynucleotide. Separation of the strands by the pore may then separate the protecting molecule from the part and reveal the part for attachment.

The part of the preceding target polynucleotide may be protected by hybridisaton to a protecting polynucleotide. The protecting polynucleotide may be separated from the part as the target polynucleotide moves through the pore. The protecting polynucleotide may form part of the target polynucleotide as discussed in more detail below. The protecting polynucleotide may be a separate polynucleotide. As discussed in more detail below, the protecting polynucleotide may protect the part from hybridisation to a part of the subsequent target polynucleotide. Alternatively, the protecting polynucleotide may prevent the action of a single strand ligase on the part. The presence protecting polynucleotide (forming a double stranded region with the part) means the ligase cannot function. Release of the protecting polynucleotide would reveal the part as a substrate for the ligase.

A part of the subsequent target polynucleotide preferably selectively hybridises to a part of the preceding polynucleotide, e.g. the site revealed on the preceding target polynucleotide as a result of its interaction with the pore is a nucleotide sequence that is complementary to a nucleotide sequence on the subsequent polynucleotide. For instance, a part of the second target polynucleotide preferably selectively hybridises to a part of the first polynucleotide. A part of the third target polynucleotide preferably selectively hybridises to a part of the second target polynucleotide and so on.

The part of the preceding target polynucleotide is preferably initially protected from hybridisation to the part of the subsequent target polynucleotide and is revealed for hybridisation as the preceding target polynucleotide moves through the pore. For instance, the part of the second target polynucleotide is preferably initially protected from hybridisation to the part of the first target polynucleotide and is revealed for hybridisation as the first target polynucleotide moves through the pore. The part of the third target polynucleotide is preferably initially protected from hybridisation to the part of the second target polynucleotide and is revealed for hybridisation as the second target polynucleotide moves through the pore and so on.

The part of the preceding target polynucleotide preferably specifically hybridises to the part of the subsequent target polynucleotide. The parts of the target polynucleotides specifically hybridise when they hybridise with preferential or high affinity to each other but do not substantially hybridise, do not hybridise or hybridise with only low affinity to other polynucleotides or sequences. The part of the preceding target polynucleotide specifically hybridises to the part of the subsequent target polynucleotide if it hybridises to the part of the subsequent target polynucleotide with a melting temperature (T_(m)) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C. or at least 10° C., greater than its T_(m) for other sequences. More preferably, the part of the preceding target polynucleotide hybridises to the part of the subsequent target polynucleotide with a T_(m) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., at least 30° C. or at least 40° C., greater than its T_(m) for other sequences. Preferably, the part of the preceding target polynucleotide hybridises to the part of the subsequent target polynucleotide with a T_(m) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., at least 30° C. or at least 40° C., greater than its T_(m) for a sequence which differs from the part of the subsequent target polynucleotide by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The part of the preceding target polynucleotide typically hybridises to the part of the subsequent target polynucleotide with a T_(m) of at least 90° C., such as at least 92° C. or at least 95° C. T_(m) can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available T_(m) calculators, such as those available over the internet.

Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1% SDS (sodium dodecyl sulfate) at 37° C. followed by a 20 wash in from 1× (0.1650 M Na⁺) to 2× (0.33 M Na⁺) SSC (standard sodium citrate) at 50° C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by a wash in from 0.5× (0.0825 M Na⁺) to 1× (0.1650 M Na⁺) SSC at 55° C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37° C., followed by a wash in 0.1× (0.0165 M Na⁺) SSC at 60° C.

The part of the preceding target polynucleotide is preferably substantially complementary to the part of the subsequent target polynucleotide. The part of the preceding target polynucleotide may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches across a region of 5, 10, 15, 20, 21, 22, 30, 40 or 50 nucleotides compared with the part of the subsequent target polynucleotide. The part of the preceding target polynucleotide is preferably complementary to the part of the subsequent target polynucleotide.

Each part is typically 50 nucleotides or fewer, such as 40 nucleotides or fewer, 30 nucleotides or fewer, 20 nucleotides or fewer, 12 nucleotides or fewer, 10 nucleotides or fewer or 5 nucleotides or fewer. Each part is typically at least 4 nucleotides in length, such as at least 5 nucleotides, at least 10 nucleotides or at least 12 nucleotides in length.

Each target polynucleotide may comprise the same parts, one which selectively hybridises to a part of the preceding polynucleotide (when it is the subsequent target polynucleotide) and one which selectively hybridises to a part of the subsequent polynucleotide (when it is the preceding polynucleotide). In such embodiment, the two parts in each target polynucleotide must be protected from one another so that they do not attach to each other and only attach to a different target polynucleotide. The parts may be protected from one another in any of the ways discussed above.

In one embodiment, the part of the preceding, e.g. first, polynucleotide that is revealed for attachment to the subsequent, e.g. second, polynucleotide is at, or close to, the opposite end of the preceding polynucleotide to the end that is fed into the pore. Where the polynucleotide passes through the pore in a 5′ to 3′ direction, the part of the preceding polynucleotide that attaches to the subsequent polynucleotide is at, or close to, the 3′ end of the preceding polynucleotide. The part of the subsequent polynucleotide that attaches to the preceding polynucleotide is then at, or close to, the 5′ end of the subsequent polynucleotide. A part close to the end of a polynucleotide is typically within, for example, 1 to 30 bases of the end of the polynucleotide, such as within 2 to 25, 3 to 20, 4 to 15 or 5 to 10 bases from the end of the polynucleotide.

In a preferred embodiment, the preceding target polynucleotide is double stranded, the part (attachment site) of the preceding target polynucleotide is in one strand (a first strand) and is hybridised to the other strand (a second strand), and the part (attachment site) is revealed for hybridisation as the two (first and second) strands separate as the preceding target polynucleotide moves through the pore. In this embodiment, the other (second) strand of the target polynucleotide is the protecting molecule. If the second strand moves through the pore, the two strands separate and the part (attachment site on the first strand) is revealed for hybridisation to the part (attachment site) on the subsequent target polynucleotide. A specific version of this embodiment is shown in FIG. 1.

The one (first) strand in the preceding target polynucleotide preferably forms a loop structure at its end. The part (attachment site) of the preceding target polynucleotide which hybridises to the part (attachment site) of the subsequent target polynucleotide is preferably adjacent to, but not part of the loop structure. In such an embodiment, hybridisation of the part in the subsequent target polynucleotide to the part in the preceding target polynucleotide elongates the loop. This embodiment results in the preceding and subsequent target polynucleotides being attached by a loop which may dehybridise and move through the pore. A specific version of this embodiment is shown in FIG. 1.

In one embodiment, both strands of the double stranded preceding target polynucleotide are preferably linked at one end by a hairpin loop. Hairpin loops are discussed in more detail below. If the two strands are linked, the two strands separate, the part is revealed for hybridisation to the part on subsequent target polynucleotide, the hairpin moves through the pore and then the strand comprising the part attached to the subsequent target polynucleotide moves through the pore.

The other (second) strand at the other end of the preceding target polynucleotide preferably comprises a leader sequence which preferentially threads into the pore. This ensures that the other (second) strand enters the pore and the part (attachment site) is revealed for hybridisation as the two strands separate. Suitable leader sequences are discussed in more detail below.

The subsequent target polynucleotide is preferably attached to the one (first) strand at the other end of the preceding target polynucleotide. As the other (second) strand, which preferably comprises a leader sequence, moves through the pore, the part (attachment site) on the one (first) strand is preferably revealed for attachment or hybridisation to the part (attachment site) on the subsequent target polynucleotide.

The subsequent target polynucleotide is preferably double stranded. In one embodiment, the two strands are preferably linked at one end by a hairpin loop. The other end of the subsequent target polynucleotide from the hairpin loop is preferably selectively attached to the preceding target polynucleotide. In particular, the other end of the subsequent target polynucleotide from the hairpin loop preferably comprises a leader sequence which is capable of selectively attaching to the preceding target polynucleotide. The free end of the leader sequence is preferably capable of hybridizing to the part of the preceding target polynucleotide. The free end of the leader sequence is preferably the part of the subsequent target polynucleotide which specifically hybridises to the part of the preceding target polynucleotide. As discussed above, the part of the preceding target polynucleotide is preferably adjacent to a loop structure in the preceding target polynucleotide.

The end of the leader sequence may be hybridised to a bridging polynucleotide which forms an overhang which is capable of selectively attaching to the preceding target polynucleotide. The overhang is preferably capable of hybridizing to the part (attachment site) of the preceding target polynucleotide. The overhang is preferably the part of the subsequent target polynucleotide which specifically hybridises to the part of the preceding target polynucleotide. In other words, the overhang comprises or constitutes the attachment site in the subsequent target polynucleotide. Hybridisation of the overhang to the part (attachment site) in the preceding target polynucleotide positions the leader of the subsequent polynucleotide adjacent to the part (attachment site) in the preceding target polynucleotide and the two parts (attachment sites) may be attached to one another. The bridging polynucleotide and/or the overhang can be any length and formed from any of the polynucleotides discussed below.

The second (subsequent) target polynucleotide is preferably selectively covalently attached to the first (preceding) target polynucleotide. For instance, in one embodiment, the part of the preceding target polynucleotide may hybridise with the part of the second target polynucleotide (e.g. where the attachment sites in the first and second polynucleotides are complementary to one another) and then the two polynucleotides may be covalently attached. Any form of covalent attachment may be used.

The subsequent (second) target polynucleotide is preferably covalently attached to the preceding (first) target polynucleotide using a ligase, a topoisomerase or by click chemistry. The ligase, topoisomerase or click chemistry results in the formation of one or more covalent bonds between the first and second target polynucleotides.

Any ligase may be used, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase. The ligase is preferably T3 DNA ligase. This commercially available, for instance from, New England BioLabs® Inc.

Any topoisomerase may be used. Suitable topopisomerases include, but are not limited to, Vaccinia DNA Topoisomerase or Human DNA Topoisomerase I.

Click chemistry is advantageous because it does not typically involve the use of enzymes. Click chemistry is a term first introduced by Kolb et al. in 2001 to describe an expanding set of powerful, selective, and modular building blocks that work reliably in both small- and large-scale applications (Kolb H C, Finn, M G, Sharpless K B, Click chemistry: diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021). They have defined the set of stringent criteria for click chemistry as follows: “The reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation. Purification if required must be by nonchromatographic methods, such as crystallization or distillation, and the product must be stable under physiological conditions”.

Suitable example of click chemistry include, but are not limited to, the following:

-   -   (a) copper-free variant of the 1,3 dipolar cycloaddition         reaction, where an azide reacts with an alkyne under strain, for         example in a cyclooctane ring;     -   (b) the reaction of an oxygen nucleophile on one linker with an         epoxide or aziridine reactive moiety on the other; and     -   (c) the Staudinger ligation, where the alkyne moiety can be         replaced by an aryl phosphine, resulting in a specific reaction         with the azide to give an amide bond.

Preferably the click chemistry reaction is the Cu (I) catalysed 1,3 dipolar cycloaddition reaction between an alkyne and an azide. In a preferred embodiment, the first group is an azide group and the second group is an alkyne group. Nucleic acid bases have already been synthesised incorporating azide and alkyne groups in preferred positions (for example Kocalka P, El-Sagheer A H, Brown T, Rapid and efficient DNA strand cross-linking by click chemistry, Chembiochem. 2008. 9(8):1280-5). Alkyne groups are available commercially from Berry Associates (Michigan, USA) and azide groups are synthesised by ATDBio.

If the preceding and subsequent polynucleotides or part thereof are modified to include groups that can form covalent bonds, the modified nucleotides are preferably offset from one another by one nucleotide in order to achieve the link. This follows the published work of Tom Brown (Kocalka et al. (2008) ChemBiochem 9 8 1280-1285).

Other preferred groups for use in the invention are shown in the following Table 1.

TABLE 1 Some preferred groups capable of forming covalent bonds Reacts Name with Structure 1,4-Bis[3-(2- pyridyldithio)propionamido]butane Thiols

1,11-bis- Maleimidotriethyleneglycol Thiols

3,3′-Dithiodipropionic acid di(N- hydroxysuccinimide ester) Primary amines

Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) Primary amines

4,4′-Diisothiocyanatostilbene-2,2′- disulfonic acid disodium salt Primary amines

Bis[2-(4- azidosalicylamido)ethyl]disulfide Photo- activated, non- specific

3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester Thiols, primary amines

4-Maleimidobutyric acid N- hydroxysuccinimide ester Thiols, primary amines

Iodoacetic acid N- hydroxysuccinimide ester Thiols, primary amines

S-Acetylthioglycolic acid N- hydroxysuccinimide ester Thiols, primary amines

Azide-PEG-maleimide Thiols, alkyne

Alkyne-PEG-maleimide Thiols, azide

Copper free click chemistry can be used in the invention because of its desirable properties. For example, it is fast, clean and not poisonous towards proteins. A good example of this is maleimide or iodoacetamide linking with a cyclooctyne functional group (DIBO). However, other suitable bio-orthogonal chemistries include, but are not limited to, Staudinger chemistry, hydrazine or hydrazide/aldehyde or ketone reagents (HyNic+4FB chemistry, including all Solulink™ reagents), Diels-Alder reagent pairs and boronic acid/salicyhydroxamate reagents.

Preferably the reactive groups are azide and hexynl groups such as 3 AzideN and 5′-hexynl-G.

Preferred pairs of non-covalent reactive groups include, but are not limited to, (i) Ni-NTA and polyhistidine, such as 6×His, and (ii) cyclodextrin and adamantine.

Polynucleotide

The two more target polynucleotides may be any type of polynucleotide. A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the polynucleotide can be oxidised or methylated. One or more nucleotides in the polynucleotide may be damaged. For instance, the polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light and are the primary cause of skin melanomas. One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. Suitable labels are described below. The polynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside.

The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably a deoxyribose.

The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. The nucleotide may comprise more than three phosphates, such as 4 or 5 phosphates. Phosphates may be attached on the 5′ or 3′ side of a nucleotide. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate. The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.

A nucleotide may be abasic (i.e. lack a nucleobase). A nucleotide may also lack a nucleobase and a sugar (i.e. is a C3 spacer).

The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide can comprise one strand of RNA hybridised to one strand of DNA. The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains. The PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone is composed of repeating glycol units linked by phosphodiester bonds. The TNA backbone is composed of repeating threose sugars linked together by phosphodiester bonds. LNA is formed from ribonucleotides as discussed above having an extra bridge connecting the 2′ oxygen and 4′ carbon in the ribose moiety.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) or deoxyribonucleic acid (DNA).

The polynucleotide can be any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs in length or 100000 or more nucleotides or nucleotide pairs in length.

Sample

The target polynucleotides may be present in any suitable sample. The sample may be a biological sample. The invention may be carried out in vitro using at least one sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. The invention may be carried out in vitro on at least one sample obtained from or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs. Alternatively, the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, broccoli or cotton.

The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

The sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below −70° C.

Membrane

The polynucleotides are contacted with a transmembrane pore in a membrane. Any membrane may be used in accordance with the invention. Suitable membranes are well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer sub-units are polymerised together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit. However, a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess. Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in aqueous media. In this case, the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane. The block copolymer may be a diblock (consisting of two monomer sub-units), but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphiphiles. The copolymer may be a triblock, tetrablock or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are constructed such that the lipid forms a monolayer membrane. These lipids are generally found in extremophiles that survive in harsh biological environments, thermophiles, halophiles and acidophiles. Their stability is believed to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating a triblock polymer that has the general motif hydrophilic-hydrophobic-hydrophilic. This material may form monomeric membranes that behave similarly to lipid bilayers and encompass a range of phase behaviours from vesicles through to laminar membranes. Membranes formed from these triblock copolymers hold several advantages over biological lipid membranes. Because the triblock copolymer is synthesised, the exact construction can be carefully controlled to provide the correct chain lengths and properties required to form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are not classed as lipid sub-materials; for example a hydrophobic polymer may be made from siloxane or other non-hydrocarbon based monomers. The hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples. This head group unit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical and environmental stability compared with biological lipid membranes, for example a much higher operational temperature or pH range. The synthetic nature of the block copolymers provides a platform to customise polymer based membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed in International Application No. PCT/GB2013/052766 (published as WO 2014/064443) or PCT/GB2013/052767 (published as WO 2014/064444).

The amphiphilic molecules may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported. The amphiphilic layer may be concave. The amphiphilic layer may be suspended from raised pillars such that the peripheral region of the amphiphilic layer (which is attached to the pillars) is higher than the amphiphilic layer region. This may allow the microparticle to travel, move, slide or roll along the membrane as described above.

Amphiphilic membranes are typically naturally mobile, essentially acting as two dimensional fluids with lipid diffusion rates of approximately 10⁻⁸ cm s-1. This means that the pore and coupled polynucleotide can typically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International Application No. PCT/GB08/000563 (published as WO 2008/102121), International Application No. PCT/GB08/004127 (published as WO 2009/077734) and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface. The lipid is normally added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent has evaporated, the solution/air interfaces on either side of the aperture are physically moved up and down past the aperture until a bilayer is formed. Planar lipid bilayers may be formed across an aperture in a membrane or across an opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore insertion. Other common methods of bilayer formation include tip-dipping, painting bilayers and patch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (for example, a pipette tip) onto the surface of a test solution that is carrying a monolayer of lipid. Again, the lipid monolayer is first generated at the solution/air interface by allowing a drop of lipid dissolved in organic solvent to evaporate at the solution surface. The bilayer is then formed by the Langmuir-Schaefer process and requires mechanical automation to move the aperture relative to the solution surface.

For painted bilayers, a drop of lipid dissolved in organic solvent is applied directly to the aperture, which is submerged in an aqueous test solution. The lipid solution is spread thinly over the aperture using a paintbrush or an equivalent. Thinning of the solvent results in the formation of a lipid bilayer. However, complete removal of the solvent from the bilayer is difficult and consequently the bilayer formed by this method is less stable and more prone to noise during electrochemical measurement.

Patch-clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by suction and a patch of the membrane becomes attached over the aperture. The method has been adapted for producing lipid bilayers by clamping liposomes which then burst to leave a lipid bilayer sealing over the aperture of the pipette. The method requires stable, giant and unilamellar liposomes and the fabrication of small apertures in materials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

In a preferred embodiment, the lipid bilayer is formed as described in International Application No. PCT/GB08/004127 (published as WO 2009/077734). Advantageously in this method, the lipid bilayer is formed from dried lipids. In a most preferred embodiment, the lipid bilayer is formed across an opening as described in WO2009/077734 (PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The two layers of lipids are arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer. The bilayer may be present in a number of lipid phases including, but not limited to, the liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipid composition is chosen such that a lipid bilayer having the required properties, such as surface charge, ability to support membrane proteins, packing density or mechanical properties, is formed. The lipid composition can comprise one or more different lipids. For instance, the lipid composition can contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may comprise naturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammonium-Propane (TAP). Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic (n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9-Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary. The length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester. The lipids may be mycolic acid.

The lipids can also be chemically-modified. The head group or the tail group of the lipids may be chemically-modified. Suitable lipids whose head groups have been chemically-modified include, but are not limited to, PEG-modified lipids, such as 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]; functionalised PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as 1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as 1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine; deuterated lipids, such as 1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked lipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. The lipids may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.

The amphiphilic layer, for example the lipid composition, typically comprises one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigmasterol; lysophospholipids, such as 1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane is a solid state layer. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as HfO₂, Si₃N₄, Al₂O₃, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid state layer may be by atomic layer deposition (ALD). The ALD solid state layer may comprise alternating layers of HfO₂ and Al₂O₃. The solid state layer may be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick. Suitable graphene layers are disclosed in International Application No. PCT/US2008/010637 (published as WO 2009/035647). Yusko et al., Nature Nanotechnology, 2011; 6: 253-260 and US Patent Application No. 2013/0048499 describe the delivery of proteins to transmembrane pores in solid state layers without the use of microparticles. The method of the invention may be used to improve the delivery in the methods disclosed in these documents.

The method is typically carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally-occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The method of the invention is typically carried out in vitro.

Transmembrane Pore

A transmembrane pore is a structure that crosses the membrane to some degree. Typically, a transmembrane pore comprises a first opening and a second opening with a lumen extending between the first opening and the second opening. The transmembrane pore permits hydrated ions driven by an applied potential to flow across or within the membrane. The transmembrane pore typically crosses the entire membrane so that hydrated ions may flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to cross the membrane. It may be closed at one end. For instance, the pore may be a well, gap, channel, trench or slit in the membrane along which or into which hydrated ions may flow.

Any transmembrane pore may be used in the invention. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores and solid state pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936).

The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a collection of polypeptides that permits hydrated ions, such as polynucleotide, to flow from one side of a membrane to the other side of the membrane. In the present invention, the transmembrane protein pore is capable of forming a pore that permits hydrated ions driven by an applied potential to flow from one side of the membrane to the other. The transmembrane protein pore preferably permits polynucleotides to flow from one side of the membrane, such as a triblock copolymer membrane, to the other. The transmembrane protein pore allows a polynucleotide, such as DNA or RNA, to be moved through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The pore is preferably made up of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 subunits. The pore is preferably a hexameric, heptameric, octameric or nonameric pore. The pore may be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β barrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typically comprises amino acids that facilitate interaction with s, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located near a constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention can be derived from β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) and other pores, such as lysenin. α-helix bundle pores comprise a barrel or channel that is formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from lysenin. Suitable pores derived from CsgG are disclosed in International Application No. PCT/EP2015/069965. Suitable pores derived from lysenin are disclosed in International Application No. PCT/GB2013/050667 (published as WO 2013/153359). The transmembrane pore may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Spl and haemolytic protein fragaceatoxin C (FraC). The wild type α-hemolysin pore is formed of 7 identical monomers or sub-units (i.e., it is heptameric). The sequence of one monomer or sub-unit of α-hemolysin-NN is shown in SEQ ID NO: 4.

The transmembrane protein pore is preferably derived from Msp, more preferably from MspA. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be a homo-oligomeric pore derived from Msp comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from Msp comprising at least one monomer that differs from the others. Preferably the pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp typically comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into membranes, such as amphiphilic layers. For example, subunits may be suspended in a purified form in a solution containing a triblock copolymer membrane such that it diffuses to the membrane and is inserted by binding to the membrane and assembling into a functional state. Alternatively, subunits may be directly inserted into the membrane using the “pick and place” method described in M. A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to that sequence based on amino acid similarity or identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid similarity or identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid similarity or identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Similarity can be measured using pairwise identity or by applying a scoring matrix such as BLOSUM62 and converting to an equivalent identity. Since they represent functional rather than evolved changes, deliberately mutated positions would be masked when determining homology. Similarity may be determined more sensitively by the application of position-specific scoring matrices using, for example, PSIBLAST on a comprehensive database of protein sequences. A different scoring matrix could be used that reflect amino acid chemico-physical properties rather than frequency of substitution over evolutionary time scales (e.g. charge).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared with MspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7. In particular, the variant may comprise the following substitution present in MspB: A138P. The variant may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant may comprise combinations of one or more of the mutations and substitutions from Msp B, C and D. The variant preferably comprises the mutation L88N. A variant of SEQ ID NO: 2 has the mutation L88N in addition to all the mutations of MS-B1 and is called MS-(B2)8. The pore used in the invention is preferably MS-(B2)8. The variant of SEQ ID NO: 2 preferably comprises one or more of D56N, D56F, E59R, G75S, G77S, A96D and Q126R. A variant of SEQ ID NO: 2 has the mutations G75S/G77S/L88N/Q126R in addition to all the mutations of MS-B1 and is called MS-B2C. The pore used in the invention is preferably MS-(B2)8 or MS-(B2C)8. The variant of SEQ ID NO: 2 preferably comprises N93D. The variant more preferably comprises the mutations G75S/G77S/L88N/N93D/Q126R.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid.

The transmembrane protein pore is preferably derived from CsgG, more preferably from CsgG from E. coli Str. K-12 substr. MC4100. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10 monomers derived from CsgG. The pore may be a homo-oligomeric pore derived from CsgG comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from CsgG comprising at least one monomer that differs from the others.

A monomer derived from CsgG typically comprises the sequence shown in SEQ ID NO: 27 or a variant thereof. A variant of SEQ ID NO: 27 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 27 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art as discussed above.

Over the entire length of the amino acid sequence of any one of SEQ ID NO: 27, a variant will preferably be at least 50% homologous to that sequence based on amino acid similarity or identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid similarity or identity to the amino acid sequence of SEQ ID NO: 27 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid similarity or identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids (“hard homology”). Homology can be measured as discussed above.

The variant of SEQ ID NO: 27 may comprise any of the mutations disclosed in International Application No. PCT/GB2015/069965 (published as WO 2016/034591). The variant of SEQ ID NO: 27 preferably comprises one or more of the following (i) one or more mutations at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192, such as one or more mutations at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, E101, E131 and T150 or N40, D43, E44, E101 and E131; (ii) mutations at Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56; (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more of the following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57. The variant may comprise any combination of (i) to (xi). If the variant comprises any one of (i) and (iii) to (xi), it may further comprise a mutation at one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

Preferred variants of SEQ ID NO: 27 which form pores in which fewer nucleotides contribute to the current as the polynucleotide moves through the pore comprise Y51A/F56A, Y51A/F56N, Y51I/F56A, Y51L/F56A, Y51T/F56A, Y51I/F56N, Y51L/F56N or Y51T/F56N or more preferably Y51I/F56A, Y51L/F56A or Y51T/F56A.

Preferred variants of SEQ ID NO: 27 which form pores displaying an increased range comprise mutations at the following positions:

Y51, F56, D149, E185, E201 and E203;

N55 and F56;

Y51 and F56;

Y51, N55 and F56; or

F56 and N102.

Preferred variants of SEQ ID NO: 27 which form pores displaying an increased range comprise:

Y51N, F56A, D149N, E185R, E201N and E203N;

N55S and F56Q;

Y51A and F56A;

Y51A and F56N;

Y51I and F56A;

Y51L and F56A;

Y51T and F56A;

Y51I and F56N;

Y51L and F56N;

Y51T and F56N;

Y51T and F56Q;

Y51A, N55S and F56A;

Y51A, N55S and F56N;

Y51T, N55S and F56Q; or

F56Q and N102R.

Preferred variants of SEQ ID NO: 27 which form pores in which fewer nucleotides contribute to the current as the polynucleotide moves through the pore comprise mutations at the following positions:

N55 and F56, such as N55X and F56Q, wherein X is any amino acid; or

Y51 and F56, such as Y51X and F56Q, wherein X is any amino acid.

Preferred variants of SEQ ID NO: 27 which form pores displaying an increased throughput comprise mutations at the following positions:

D149, E185 and E203;

D149, E185, E201 and E203; or

D149, E185, D195, E201 and E203.

Preferred variants which form pores displaying an increased throughput comprise:

D149N, E185N and E203N;

D149N, E185N, E201N and E203N;

D149N, E185R, D195N, E201N and E203N; or

D149N, E185R, D195N, E201R and E203N.

Preferred variants of SEQ ID NO: 7 which form pores in which capture of the polynucleotide is increased comprise the following mutations:

D43N/Y51T/F56Q;

E44N/Y51T/F56Q;

D43N/E44N/Y51T/F56Q;

Y51T/F56Q/Q62R;

D43N/Y51T/F56Q/Q62R;

E44N/Y51T/F56Q/Q62R; or

D43N/E44N/Y51T/F56Q/Q62R.

Preferred variants of SEQ ID NO: 27 comprise the following mutations:

D149R/E185R/E201R/E203R or Y51T/F56Q/D149R/E185R/E201R/E203R;

D149N/E185N/E201N/E203N or Y51T/F56Q/D149N/E185N/E201N/E203N;

E201R/E203R or Y51T/F56Q/E201R/E203R

E201N/E203R or Y51T/F56Q/E201N/E203R;

E203R or Y51T/F56Q/E203R;

E203N or Y51T/F56Q/E203N;

E201R or Y51T/F56Q/E201R;

E201N or Y51T/F56Q/E201N;

E185R or Y51T/F56Q/E185R;

E185N or Y51T/F56Q/E185N;

D149R or Y51T/F56Q/D149R;

D149N or Y51T/F56Q/D149N;

R142E or Y51T/F56Q/R142E;

R142N or Y51T/F56Q/R142N;

R192E or Y51T/F56Q/R192E; or

R192N or Y51T/F56Q/R192N.

Preferred variants of SEQ ID NO: 27 comprise the following mutations:

Y51A/F56Q/E101N/N102R;

Y51A/F56Q/R97N/N102G;

Y51A/F56Q/R97N/N102R;

Y51A/F56Q/R97N;

Y51A/F56Q/R97G;

Y51A/F56Q/R97L;

Y51A/F56Q/N102R;

Y51A/F56Q/N102F;

Y51A/F56Q/N102G;

Y51A/F56Q/E101R;

Y51A/F56Q/E101F;

Y51A/F56Q/E101N; or

Y51A/F56Q/E101G

The variant of SEQ ID NO: 27 may comprise any of the substitutions present in another CsgG homologue. Preferred CsgG homologues are shown in SEQ ID NOs: 3 to 7 and 26 to 41 of International Application No. PCT/GB2015/069965 (published as WO 2016/034591).

Any of the proteins described herein, such as the transmembrane protein pores, may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore or construct. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

The pore may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the transmembrane protein pores, may be made synthetically or by recombinant means. For example, the pore may be synthesised by in vitro translation and transcription (IVTT). The amino acid sequence of the pore may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When a protein is produced by synthetic means, such amino acids may be introduced during production. The pore may also be altered following either synthetic or recombinant production.

Any of the proteins described herein, such as the transmembrane protein pores, can be produced using standard methods known in the art. Polynucleotide sequences encoding a pore or construct may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or construct may be expressed in a bacterial host cell using standard techniques in the art. The pore may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The pore may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.

Microparticle

A microparticle may be used to deliver the target polynucleotide to the transmembrane pore. Any number of microparticles can be used in the method of the invention. For instance, the method of the invention may use a single microparticle or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 1,000, 5,000, 10,000, 100,000, 500,000 or 1,000,000 or more microparticles. If two or more microparticles are used, the microparticles may be the same. Alternatively, a mixture of different microparticles may be used.

Each microparticle may have one polynucleotide attached. Alternatively, each microparticle may have two or more polynucleotides, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 100,000 or more, 1000,000 or more or 5000,000 or more polynucleotides, attached. A microparticle may be substantially or completed coated or covered with polynucleotide. A microparticle may have a polynucleotide attached over substantially all of or all of its surface. A microparticle may be attached to a polynucleotide via an adaptor. The adaptor may be a Y-adaptor or a hairpin adaptor (see below)

A polynucleotide, i.e. a single instance of an polynucleotide, may be attached to two or more microparticles. A polynucleotide, i.e. a single instance of an polynucleotide, may be attached to any number of the microparticles discussed above.

A microparticle is a microscopic particle whose size is typically measured in micrometres (μm). Microparticles may also known as microspheres or microbeads. The microparticle may be a nanoparticle. A nanoparticle is a microscopic particle whose size is typically measured in nanometres (nm).

A microparticle typically has a particle size of from about 0.001 μm to about 500 μm. For instance, a nanoparticle may have a particle size of from about 0.01 μm to about 200 μm or about 0.1 μm to about 100 μm. More often, a microparticle has a particle size of from about 0.5 μm to about 100 μm, or for instance from about 1 μm to about 50 μm. The microparticle may have a particle size of from about 1 nm to about 1000 nm, such as from about 10 nm to about 500 nm, about 20 nm to about 200 nm or from about 30 nm to about 100 nm.

A microparticle may be spherical or non-spherical. Spherical microparticles may be called microspheres. Non-spherical particles may for instance be plate-shaped, needle-shaped, irregular or tubular. The term “particle size” as used herein means the diameter of the particle if the particle is spherical or, if the particle is non-spherical, the volume-based particle size. The volume-based particle size is the diameter of the sphere that has the same volume as the non-spherical particle in question.

If two or more microparticles are used in the method, the average particle size of the microparticles may be any of the sizes discussed above, such as from about 0.5 μm to about 500 μm. A population of two or more microparticles preferably has a coefficient of variation (ratio of the standard deviation to the mean) of 10% or less, such as 5% or less or 2% or less.

Any method may be used to determine the size of the microparticle. Suitable methods include, but are not limited to, flow cytometry (see, for example, Chandler et al., J Thromb Haemost. 2011 June; 9(6):1216-24).

The microparticle may be formed from any material. The microparticle is preferably formed from a ceramic, glass, silica, a polymer or a metal. The polymer may be a natural polymer, such as polyhydroxyalkanoate, dextran, polylactide, agarose, cellulose, starch or chitosan, or a synthetic polymer, such as polyurethane, polystyrene, poly(vinyl chloride), silane or methacrylate. Suitable microparticles are known in the art and are commercially available. Ceramic and glass microspheres are commercially available from 3M®. Silica and polymer microparticles are commercially available from EPRUI Nanoparticles & Microspheres Co. Ltd. Microparticles are also commercially available from Polysciences Inc., Bangs Laboratories Inc. and Life Technologies.

The microparticle may be solid. The microparticle may be hollow. The microparticle may be formed from polymer fibers.

The microparticle may be derived from the kit used to extract and isolate the polynucleotide.

The surface of the microparticle may interact with and attach the polynucleotide. The surface may naturally interact with the polynucleotide without functionalisation. The surface of the microparticle is typically functionalised to facilitate attachment of the polynucleotide. Suitable functionalisations are known in the art. For instance, the surface of the microparticle may be functionalised with a polyhistidine-tag (hexa histidine-tag, 6×His-tag, His6 tag or His-tag®), Ni-NTA, streptavidin, biotin, an oligonucleotide, a polynucleotide (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipid, FLAG-tag (FLAG octapeptide, polynucleotide binding proteins (including any of those discussed below), peptides, proteins, antibodies or antibody fragments. Antibody fragments are discussed in more detail below. The microparticle may also be functionalised with any of the linkers or groups discussed below with reference to attachment.

The microparticle may be functionalised with a molecule or group which specifically binds to the polynucleotide. In this instance, the polynucleotide which will be attached to the microparticle and delivered to the transmembrane pore may be called the target polynucleotide. This allows the microparticle to select or capture the target polynucleotide from a sample containing other polynucleotides. A molecule or group specifically binds to the target polynucleotide if it binds to the target polynucleotide with preferential or high affinity, but does not bind or binds with only low affinity to other or different polynucleotides. A molecule or group binds with preferential or high affinity if it binds with a Kd of 1×10⁻⁶ M or less, more preferably 1×10⁻⁷ M or less, 5×10⁻⁸M or less, more preferably 1×10⁻⁸M or less or more preferably 5×10⁻⁹M or less. A molecule or group binds with low affinity if it binds with a Kd of 1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably 1×10⁻⁴ M or more, more preferably 1×10⁻³M or more, even more preferably 1×10⁻² M or more.

Preferably, the molecule or group binds to the target polynucleotide with an affinity that is at least 10 times, such as at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000 or at least 10,000 times, greater than its affinity for other polynucleotides. Affinity can be measured using known binding assays, such as those that make use of fluorescence and radioisotopes. Competitive binding assays are also known in the art. The strength of binding between peptides or proteins and polynucleotides can be measured using nanopore force spectroscopy as described in Hornblower et al., Nature Methods. 4: 315-317. (2007).

The microparticle may be functionalised with an oligonucleotide or a polynucleotide (such as any of those discussed above) which specifically hybridises to the target polynucleotide or comprises a portion or region which is complementary to a portion or region of the target polynucleotide. This allows the microparticle to select or capture the target polynucleotide from a sample containing other polynucleotides. An oligonucleotide or polynucleotide specifically hybridises to a target polynucleotide when it hybridises with preferential or high affinity to the target polynucleotide but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other polynucleotide. An oligonucleotide or polynucleotide specifically hybridises if it hybridises to the target polynucleotide with a melting temperature (T_(m)) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C. or at least 10° C., greater than its T_(m) for other sequences. More preferably, the oligonucleotide or polynucleotide hybridises to the target polynucleotide with a T_(m) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., at least 30° C. or at least 40° C., greater than its T_(m) for other nucleic acids. Preferably, the oligonucleotide or polynucleotide hybridises to the target polynucleotide with a T_(m) that is at least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., at least 30° C. or at least 40° C., greater than its T_(m) for a sequence which differs from the target polynucleotide by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The oligonucleotide or polynucleotide typically hybridises to the target polynucleotide with a T_(m) of at least 90° C., such as at least 92° C. or at least 95° C. T_(m) can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available T_(m) calculators, such as those available over the internet.

Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1% SDS (sodium dodecyl sulfate) at 37° C. followed by a 20 wash in from 1× (0.1650 M Na⁺) to 2× (0.33 M Na⁺) SSC (standard sodium citrate) at 50° C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by a wash in from 0.5× (0.0825 M Na⁺) to 1× (0.1650 M Na⁺) SSC at 55° C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37° C., followed by a wash in 0.1× (0.0165 M Na⁺) SSC at 60° C.

The oligonucleotide or polynucleotide may comprise a portion or region which is substantially complementary to a portion or region of the target polynucleotide. The region or portion of the oligonucleotide or polynucleotide may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches across a region of 5, 10, 15, 20, 21, 22, 30, 40 or 50 nucleotides compared with the portion or region in the target polynucleotide.

A portion of region is typically 50 nucleotides or fewer, such as 40 nucleotides or fewer, 30 nucleotides or fewer, 20 nucleotides or fewer, 10 nucleotides or fewer or 5 nucleotides or fewer.

The microparticle is preferably paramagnetic or magnetic. The microparticle preferably comprises a paramagnetic or a superparamagnetic material or a paramagnetic or a superparamagnetic metal, such as iron. Any suitable magnetic microparticle may be used. For instance, magnetic beads commercially available from, for instance, Clontech, Promega, Invitrogen ThermoFisher Scientific and NEB, may be used. In some embodiments, the microparticle comprises a magnetic particle with an organic group such as a metal-chelating group, such as nitrilotriacetic acid (NTA), attached. The organic component may, for instance, comprise a group selected from —C(═O)O—, —C—O—C—, —C(═O)—, —NH—, —C(═O)—NH, —C(═O)—CH₂—I, —S(═O)₂— and —S—. The organic component may comprise a metal chelating group, such as NTA (nitrilotriacetic acid). Usually, a metal such as gold, iron, nickel or cobalt is also attached to the metal-chelating group. Magnetic beads of this sort are commonly used for capturing His-tagged proteins, but are also suitable for use in the invention.

The microparticle is most preferably a His-Tag Dynabead® which is commercially available from Life Technologies, Mag Strep beads from IBA, Streptavidin magnetic beads from NEB, Solid Phase Reversible Immobilization (SPRI) beads or Agencourt AMPure XP beads from Beckman Coulter or Dynabeads® MyOne™ Streptavidin C1 (ThermoFisher Scientific).

Coupling

The first target polynucleotide and/or the one or more subsequent target polynucleotides preferably comprise one or more anchors which are capable of coupling to the membrane. The method preferably further comprises coupling the target polynucleotide to the membrane using the one or more anchors.

The anchor comprises a group which couples (or binds) to the polynucleotide and a group which couples (or binds) to the membrane. Each anchor may covalently couple (or bind) to the polynucleotide and/or the membrane.

The polynucleotide may be coupled to the membrane using any number of anchors, such as 2, 3, 4 or more anchors. For instance, the polynucleotide may be coupled to the membrane using two anchors each of which separately couples (or binds) to both the polynucleotide and membrane.

The one or more anchors may comprise one or more molecular brakes. Each anchor may comprise one or more molecular brakes. The molecular brake(s) may be any of those discussed below.

If the membrane is an amphiphilic layer, such as a triblock copolymer membrane, the one or more anchors preferably comprise a polypeptide anchor present in the membrane and/or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid, for example cholesterol, palmitate or tocopherol. In preferred embodiments, the one or more anchors are not the pore.

The components of the membrane, such as the amphiphilic molecules, copolymer or lipids, may be chemically-modified or functionalised to form the one or more anchors. Examples of suitable chemical modifications and suitable ways of functionalising the components of the membrane are discussed in more detail below. Any proportion of the membrane components may be functionalised, for example at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or 100%.

The polynucleotide may be coupled directly to the membrane. The one or more anchors used to couple the polynucleotide to the membrane preferably comprise a linker. The one or more anchors may comprise one or more, such as 2, 3, 4 or more, linkers. One linker may be used to couple more than one, such as 2, 3, 4 or more, polynucleotides to the membrane.

Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs), polysaccharides and polypeptides. These linkers may be linear, branched or circular. For instance, the linker may be a circular polynucleotide. The polynucleotide may hybridise to a complementary sequence on the circular polynucleotide linker.

The one or more anchors or one or more linkers may comprise a component that can be cut or broken down, such as a restriction site or a photolabile group.

Functionalised linkers and the ways in which they can couple molecules are known in the art. For instance, linkers functionalised with maleimide groups will react with and attach to cysteine residues in proteins. In the context of this invention, the protein may be present in the membrane, may be the polynucleotide itself or may be used to couple (or bind) to the polynucleotide. This is discussed in more detail below.

Crosslinkage of polynucleotides can be avoided using a “lock and key” arrangement. Only one end of each linker may react together to form a longer linker and the other ends of the linker each react with the polynucleotide or membrane respectively. Such linkers are described in International Application No. PCT/GB10/000132 (published as WO 2010/086602).

The use of a linker is preferred in the sequencing embodiments discussed below. If a polynucleotide is permanently coupled directly to the membrane in the sense that it does not uncouple when interacting with the pore, then some sequence data will be lost as the sequencing run cannot continue to the end of the polynucleotide due to the distance between the membrane and the pore. If a linker is used, then the polynucleotide can be processed to completion.

The coupling may be permanent or stable. In other words, the coupling may be such that the polynucleotide remains coupled to the membrane when interacting with the pore.

The coupling may be transient. In other words, the coupling may be such that the polynucleotide may decouple from the membrane when interacting with the pore. For certain applications, such as aptamer detection and polynucleotide sequencing, the transient nature of the coupling is preferred. If a permanent or stable linker is attached directly to either the 5′ or 3′ end of a polynucleotide and the linker is shorter than the distance between the membrane and the transmembrane pore's channel, then some sequence data will be lost as the sequencing run cannot continue to the end of the polynucleotide. If the coupling is transient, then when the coupled end randomly becomes free of the membrane, then the polynucleotide can be processed to completion. Chemical groups that form permanent/stable or transient links are discussed in more detail below. The polynucleotide may be transiently coupled to an amphiphilic layer or triblock copolymer membrane using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atom, such as hexadecanoic acid, may be used.

In preferred embodiments, a polynucleotide, such as a nucleic acid, is coupled to an amphiphilic layer such as a triblock copolymer membrane or lipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers has been carried out previously with various different tethering strategies. These are summarised in Table 2 below.

TABLE 2 Anchor Type of comprising coupling Reference Thiol Stable Yoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tethered vesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7. Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior of giant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68 Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant Stable van Lengerich, B., R. J. Rawle, et al. (e.g. Lipid, “Covalent attachment of lipid vesicles to a Palmitate, fluid-supported bilayer allows observation of etc) DNA-mediated vesicle interactions.” Langmuir 26(11): 8666-72

Synthetic polynucleotides and/or linkers may be functionalised using a modified phosphoramidite in the synthesis reaction, which is easily compatible for the direct addition of suitable anchoring groups, such as cholesterol, tocopherol, palmitate, thiol, lipid and biotin groups. These different attachment chemistries give a suite of options for attachment to polynucleotides. Each different modification group couples the polynucleotide in a slightly different way and coupling is not always permanent so giving different dwell times for the polynucleotide to the membrane. The advantages of transient coupling are discussed above.

Coupling of polynucleotides to a linker or to a functionalised membrane can also be achieved by a number of other means provided that a complementary reactive group or an anchoring group can be added to the polynucleotide. The addition of reactive groups to either end of a polynucleotide has been reported previously. A thiol group can be added to the 5′ of ssDNA or dsDNA using T4 polynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “A facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids.” Nucleic Acids Res 35(10): e77). An azide group can be added to the 5′-phosphate of ssDNA or dsDNA using T4 polynucleotide kinase and γ-[2-Azidoethyl]-ATP or γ-[6-Azidohexyl]-ATP. Using thiol or Click chemistry a tether, containing either a thiol, iodoacetamide OPSS or maleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne group (reactive to azides), can be covalently attached to the polynucleotide. A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tchen, et al. (1988). “Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase.” Anal Biochem 169(2): 376-82). Streptavidin/biotin and/or streptavidin/desthiobiotin coupling may be used for any other polynucleotide. The Examples below describes how a polynucleotide can be coupled to a membrane using streptavidin/biotin and streptavidin/desthiobiotin. It may also be possible that anchors may be directly added to polynucleotides using terminal transferase with suitably modified nucleotides (e.g. cholesterol or palmitate).

The one or more anchors preferably couple the polynucleotide to the membrane via hybridisation. The hybridisation may be present in any part of the one or more anchors, such as between the one or more anchors and the polynucleotide, within the one or more anchors or between the one or more anchors and the membrane. Hybridisation in the one or more anchors allows coupling in a transient manner as discussed above. For instance, a linker may comprise two or more polynucleotides, such as 3, 4 or 5 polynucleotides, hybridised together. The one or more anchors may hybridise to the polynucleotide. The one or more anchors may hybridise directly to the polynucleotide, directly to a Y adaptor and/or leader sequence attached to the polynucleotide or directly to a hairpin loop adaptor attached to the polynucleotide (as discussed in more detail below). Alternatively, the one or more anchors may be hybridised to one or more, such as 2 or 3, intermediate polynucleotides (or “splints”) which are hybridised to the polynucleotide, to a Y adaptor and/or leader sequence attached to the polynucleotide or to a hairpin loop adaptor attached to the polynucleotide (as discussed in more detail below).

The one or more anchors may comprise a single stranded or double stranded polynucleotide. One part of the anchor may be ligated to a single stranded or double stranded polynucleotide analyte. Ligation of short pieces of ssDNA have been reported using T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992). “Ligation-anchored PCR: a simple amplification technique with single-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5). Alternatively, either a single stranded or double stranded polynucleotide can be ligated to a double stranded polynucleotide and then the two strands separated by thermal or chemical denaturation. To a double stranded polynucleotide, it is possible to add either a piece of single stranded polynucleotide to one or both of the ends of the duplex, or a double stranded polynucleotide to one or both ends. For addition of single stranded polynucleotides to the double stranded polynucleotide, this can be achieved using T4 RNA ligase I as for ligation to other regions of single stranded polynucleotides. For addition of double stranded polynucleotides to a double stranded polynucleotide then ligation can be “blunt-ended”, with complementary 3′ dA/dT tails on the polynucleotide and added polynucleotide respectively (as is routinely done for many sample prep applications to prevent concatemer or dimer formation) or using “sticky-ends” generated by restriction digestion of the polynucleotide and ligation of compatible adapters. Then, when the duplex is melted, each single strand will have either a 5′ or 3′ modification if a single stranded polynucleotide was used for ligation or a modification at the 5′ end, the 3′ end or both if a double stranded polynucleotide was used for ligation.

If the polynucleotide is a synthetic strand, the one or more anchors can be incorporated during the chemical synthesis of the polynucleotide. For instance, the polynucleotide can be synthesised using a primer having a reactive group attached to it.

Adenylated polynucleotides are intermediates in ligation reactions, where an adenosine-monophosphate is attached to the 5′-phosphate of the polynucleotide. Various kits are available for generation of this intermediate, such as the 5′ DNA Adenylation Kit from NEB. By substituting ATP in the reaction for a modified nucleotide triphosphate, then addition of reactive groups (such as thiols, amines, biotin, azides, etc) to the 5′ of a polynucleotide can be possible. It may also be possible that anchors could be directly added to polynucleotides using a 5′ DNA adenylation kit with suitably modified nucleotides (e.g. cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA is using polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number of copies of the same section of DNA can be generated, where for each copy the 5′ of each strand in the duplex will be a synthetic polynucleotide. Single or multiple nucleotides can be added to 3′ end of single or double stranded DNA by employing a polymerase. Examples of polymerases which could be used include, but are not limited to, Terminal Transferase, Klenow and E. coli Poly(A) polymerase). By substituting ATP in the reaction for a modified nucleotide triphosphate then anchors, such as cholesterol, thiol, amine, azide, biotin or lipid, can be incorporated into double stranded polynucleotides. Therefore, each copy of the amplified polynucleotide will contain an anchor.

Ideally, the polynucleotide is coupled to the membrane without having to functionalise the polynucleotide. This can be achieved by coupling the one or more anchors, such as a molecular brake or a chemical group, to the membrane and allowing the one or more anchors to interact with the polynucleotide or by functionalizing the membrane. The one or more anchors may be coupled to the membrane by any of the methods described herein. In particular, the one or more anchors may comprise one or more linkers, such as maleimide functionalised linkers.

In this embodiment, the polynucleotide is typically RNA, DNA, PNA, TNA or LNA and may be double or single stranded. This embodiment is particularly suited to genomic DNA polynucleotides.

The one or more anchors can comprise any group that couples to, binds to or interacts with single or double stranded polynucleotides, specific nucleotide sequences within the polynucleotide or patterns of modified nucleotides within the polynucleotide, or any other ligand that is present on the polynucleotide.

Suitable binding proteins for use in anchors include, but are not limited to, E. coli single stranded binding protein, P5 single stranded binding protein, T4 gp32 single stranded binding protein, the TOPO V dsDNA binding region, human histone proteins, E. coli HU DNA binding protein and other archaeal, prokaryotic or eukaryotic single stranded or double stranded polynucleotide (or nucleic acid) binding proteins, including those listed below.

The specific nucleotide sequences could be sequences recognised by transcription factors, ribosomes, endonucleases, topoisomerases or replication initiation factors. The patterns of modified nucleotides could be patterns of methylation or damage.

The one or more anchors can comprise any group which couples to, binds to, intercalates with or interacts with a polynucleotide. The group may intercalate or interact with the polynucleotide via electrostatic, hydrogen bonding or Van der Waals interactions. Such groups include a lysine monomer, poly-lysine (which will interact with ssDNA or dsDNA), ethidium bromide (which will intercalate with dsDNA), universal bases or universal nucleotides (which can hybridise with any polynucleotide) and osmium complexes (which can react to methylated bases). A polynucleotide may therefore be coupled to the membrane using one or more universal nucleotides attached to the membrane. Each universal nucleotide may be coupled to the membrane using one or more linkers. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). The universal nucleotide more preferably comprises one of the following nucleosides: 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O′-methylinosine, 4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2′-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenyl C-2′-deoxyribosyl nucleoside, 2′-deoxynebularine, 2′-deoxyisoguanosine, K-2′-deoxyribose, P-2′-deoxyribose and pyrrolidine. The universal nucleotide more preferably comprises 2′-deoxyinosine. The universal nucleotide is more preferably IMP or dIMP. The universal nucleotide is most preferably dPMP (2′-Deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2,6-diaminopurine monophosphate).

The one or more anchors may couple to (or bind to) the polynucleotide via Hoogsteen hydrogen bonds (where two nucleobases are held together by hydrogen bonds) or reversed Hoogsteen hydrogen bonds (where one nucleobase is rotated through 180° with respect to the other nucleobase). For instance, the one or more anchors may comprise one or more nucleotides, one or more oligonucleotides or one or more polynucleotides which form Hoogsteen hydrogen bonds or reversed Hoogsteen hydrogen bonds with the polynucleotide. These types of hydrogen bonds allow a third polynucleotide strand to wind around a double stranded helix and form a triplex. The one or more anchors may couple to (or bind to) a double stranded polynucleotide by forming a triplex with the double stranded duplex.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50% or 100% of the membrane components may be functionalised.

Where the one or more anchors comprise a protein, they may be able to anchor directly into the membrane without further functonalisation, for example if it already has an external hydrophobic region which is compatible with the membrane. Examples of such proteins include, but are not limited to, transmembrane proteins, intramembrane proteins and membrane proteins. Alternatively the protein may be expressed with a genetically fused hydrophobic region which is compatible with the membrane. Such hydrophobic protein regions are known in the art.

The one or more anchors are preferably mixed with the polynucleotide before delivery to the membrane, but the one or more anchors may be contacted with the membrane and subsequently contacted with the polynucleotide.

In another aspect the polynucleotide may be functionalised, using methods described above, so that it can be recognised by a specific binding group. Specifically the polynucleotide may be functionalised with a ligand such as biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or a fusion protein), Ni-NTA (for binding to poly-histidine or poly-histidine tagged proteins) or peptides (such as an antigen).

According to a preferred embodiment, the one or more anchors may be used to couple a polynucleotide to the membrane when the polynucleotide is attached to a leader sequence which preferentially threads into the pore. Leader sequences are discussed in more detail below. Preferably, the polynucleotide is attached (such as ligated) to a leader sequence which preferentially threads into the pore. Such a leader sequence may comprise a homopolymeric polynucleotide or an abasic region. The leader sequence is typically designed to hybridise to the one or more anchors either directly or via one or more intermediate polynucleotides (or splints). In such instances, the one or more anchors typically comprise a polynucleotide sequence which is complementary to a sequence in the leader sequence or a sequence in the one or more intermediate polynucleotides (or splints). In such instances, the one or more splints typically comprise a polynucleotide sequence which is complementary to a sequence in the leader sequence.

Any of the methods discussed above for coupling polynucleotides to membranes, such as amphiphilic layers, can of course be applied to other polynucleotide and membrane combinations. In some embodiments, an amino acid, peptide, polypeptide or protein is coupled to an amphiphilic layer, such as a triblock copolymer layer or lipid bilayer. Various methodologies for the chemical attachment of such polynucleotides are available. An example of a molecule used in chemical attachment is EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactive groups can also be added to the 5′ of polynucleotides using commercially available kits (Thermo Pierce, Part No. 22980). Suitable methods include, but are not limited to, transient affinity attachment using histidine residues and Ni-NTA, as well as more robust covalent attachment by reactive cysteines, lysines or non natural amino acids.

Polynucleotide Characterisation

The method of the invention involves characterising the target polynucleotides. As the target polynucleotides are contacted with the pore, one or more measurements which are indicative of one or more characteristics of the target polynucleotides are taken as the concatenated polynucleotide moves with respect to the pore.

The polynucleotides can be naturally occurring or artificial. For instance, the method may be used to verify the sequence of two or more manufactured oligonucleotides. The methods are typically carried out in vitro.

The method may involve measuring two, three, four or five or more characteristics of each polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified. Any combination of (i) to (v) may be measured in accordance with the invention, such as {i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii}, {ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iv}, {i,ii,v}, {i,iii,iv}, {i,iii,v}, {ii,iii,iv}, {ii,iii,v}, {ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v}, {i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}.

For (i), the length of the polynucleotide may be measured for example by determining the number of interactions between the polynucleotide and the pore or the duration of interaction between the polynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a number of ways. The identity of the polynucleotide may be measured in conjunction with measurement of the sequence of the polynucleotide or without measurement of the sequence of the polynucleotide. The former is straightforward; the polynucleotide is sequenced and thereby identified. The latter may be done in several ways. For instance, the presence of a particular motif in the polynucleotide may be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, the measurement of a particular electrical and/or optical signal in the method may identify the polynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways. For instance, if the method involves an electrical measurement, the secondary structure may be measured using a change in dwell time or a change in current flowing through the pore. This allows regions of single-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured. The method preferably comprises determining whether or not the polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore which can be measured using the methods described below. For instance, methylcyotsine may be distinguished from cytosine on the basis of the current flowing through the pore during its interaction with each nucleotide.

The methods may be carried out using any apparatus that is suitable for investigating a membrane/pore system in which a pore is present in a membrane. The method may be carried out using any apparatus that is suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier typically has an aperture in which the membrane containing the pore is formed. Alternatively the barrier forms the membrane in which the pore is present.

The methods may be carried out using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120).

A variety of different types of measurements may be made. This includes without limitation: electrical measurements and optical measurements. A suitable optical method involving the measurement of fluorescence is disclosed by J. Am. Chem. Soc. 2009, 131 1652-1653. Possible electrical measurements include: current measurements, impedance measurements, tunnelling measurements (Ivanov A P et al., Nano Lett. 2011 Jan. 12; 11(1):279-85), and FET measurements (International Application WO 2005/124888). Optical measurements may be combined with electrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January; 81(1):014301). The measurement may be a transmembrane current measurement such as measurement of ionic current flowing through the pore.

Electrical measurements may be made using standard single channel recording equipment as describe in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and International Application WO 2000/28312. Alternatively, electrical measurements may be made using a multi-channel system, for example as described in International Application WO 2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across the membrane. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is using a salt gradient across a membrane, such as an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am Chem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the current passing through the pore as a polynucleotide moves with respect to the pore is used to estimate or determine the sequence of the polynucleotide. This is strand sequencing.

The methods may involve measuring the current passing through the pore as the polynucleotide moves with respect to the pore. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The methods may be carried out using a patch clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a current passing through the pore as the polynucleotide moves with respect to the pore. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +5 V to −5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. The voltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential.

The methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. In the exemplary apparatus discussed above, the salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is typically used. KCl, NaCl and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric across the membrane. For instance, the type and/or concentration of the charge carriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration may be 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from 150 mM to 1 M. The method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffer. The methods are typically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to 95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80° C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typically carried out at room temperature. The methods are optionally carried out at a temperature that supports enzyme function, such as about 37° C.

Molecular Brake The movement of the concatenated polynucleotide through the pore is preferably controlled by a molecular brake. The molecular brake is preferably bound to the first target polynucleotide before it is contacted with the transmembrane pore and the protein controls the movement of the entire concatenated polynucleotide through the pore. The molecular brake may be attached to a Y adaptor present on the first target polynucleotide as discussed below. The one or more subsequent polynucleotides preferably do not have a molecular brake bound to them before they are attached to the first target polynucleotide.

Any molecular brake may be used including any of those disclosed in International Application No. PCT/GB2014/052737 (published as WO 2015/110777).

The molecular brake is preferably a polynucleotide binding protein. The polynucleotide binding protein may be any protein that is capable of binding to the polynucleotide and controlling its movement through the pore. It is straightforward in the art to determine whether or not a protein binds to a polynucleotide. The protein typically interacts with and modifies at least one property of the polynucleotide. The protein may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The moiety may modify the polynucleotide by orienting it or moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that is capable of interacting with and modifying at least one property of a polynucleotide. The enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The enzyme may modify the polynucleotide by orienting it or moving it to a specific position. The polynucleotide handling enzyme does not need to display enzymatic activity as long as it is capable of binding the polynucleotide and controlling its movement through the pore. For instance, the enzyme may be modified to remove its enzymatic activity or may be used under conditions which prevent it from acting as an enzyme. Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from a nucleolytic enzyme. The polynucleotide handling enzyme used in the construct of the enzyme is more preferably derived from a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzyme may be any of those disclosed in International Application No. PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases, translocases and topoisomerases, such as gyrases. Suitable enzymes include, but are not limited to, exonuclease I from E. coli (SEQ ID NO: 11), exonuclease III enzyme from E. coli (SEQ ID NO: 13), RecJ from T. thermophilus (SEQ ID NO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17), TatD exonuclease and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 15 or a variant thereof interact to form a trimer exonuclease. The polymerase may be PyroPhage® 3173 DNA Polymerase (which is commercially available from Lucigen® Corporation), SD Polymerase (commercially available from Bioron®) or variants thereof. The enzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variant thereof. The topoisomerase is preferably a member of any of the Moiety Classification (EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme is most preferably derived from a helicase. The helicase may be or be derived from a He1308 helicase, a RecD helicase, such as TraI helicase or a TrwC helicase, a XPD helicase or a Dda helicase. The helicase may be or be derived from He1308 Mbu (SEQ ID NO: 18), He1308 Csy (SEQ ID NO: 19), He1308 Tga (SEQ ID NO: 20), He1308 Mhu (SEQ ID NO: 21), TraI Eco (SEQ ID NO: 22), XPD Mbu (SEQ ID NO: 23) or a variant thereof.

The helicase may be any of the helicases, modified helicases or helicase constructs disclosed in International Application Nos. PCT/GB2012/052579 (published as WO 2013/057495); PCT/GB2012/053274 (published as WO 2013/098562); PCT/GB2012/053273 (published as WO2013098561); PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259); PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736 (published as WO/2015/055981).

The helicase preferably comprises the sequence shown in SEQ ID NO: 25 (Trwc Cba) or as variant thereof, the sequence shown in SEQ ID NO: 18 (He1308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24 (Dda) or a variant thereof. Variants may differ from the native sequences in any of the ways discussed below for transmembrane pores. A preferred variant of SEQ ID NO: 24 comprises (a) E94C and A360C or (b) E94C, A360C, C109A and C136A and then optionally (ΔM1)G1 (i.e. deletion of M1 and then addition G1). It may also be termed M1G. Any of the variants discussed above may further comprise M1G.

The Dda helicase preferably comprises any of the modifications disclosed in International Application Nos. PCT/GB2014/052736 and PCT/GB2015/052916 (published as WO/2015/055981 and WO 2016/055777).

Any number of helicases may be used in accordance with the invention. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting the polynucleotide with two or more helicases. The two or more helicases are typically the same helicase. The two or more helicases may be different helicases.

The two or more helicases may be any combination of the helicases mentioned above. The two or more helicases may be two or more Dda helicases. The two or more helicases may be one or more Dda helicases and one or more TrwC helicases. The two or more helicases may be different variants of the same helicase.

The two or more helicases are preferably attached to one another. The two or more helicases are more preferably covalently attached to one another. The helicases may be attached in any order and using any method. Preferred helicase constructs for use in the invention are described in International Application Nos. PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259); PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

A variant of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 is an enzyme that has an amino acid sequence which varies from that of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and which retains polynucleotide binding ability. This can be measured using any method known in the art. For instance, the variant can be contacted with a polynucleotide and its ability to bind to and move along the polynucleotide can be measured. The variant may include modifications that facilitate binding of the polynucleotide and/or facilitate its activity at high salt concentrations and/or room temperature. Variants may be modified such that they bind polynucleotides (i.e. retain polynucleotide binding ability) but do not function as a helicase (i.e. do not move along polynucleotides when provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺). Such modifications are known in the art. For instance, modification of the Mg²⁺ binding domain in helicases typically results in variants which do not function as helicases. These types of variants may act as molecular brakes (see below).

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferably be at least 50% homologous to that sequence based on amino acid similarity or identity. More preferably, the variant polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid similarity or identity to the amino acid sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid similarity or identity over a stretch of 200 or more, for example 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 or more, contiguous amino acids (“hard homology”). Homology is determined as described above. The variant may differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NO: 2 and 4 above. The enzyme may be covalently attached to the pore. Any method may be used to covalently attach the enzyme to the pore.

A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with the mutation Q594A). This variant does not function as a helicase (i.e. binds polynucleotides but does not move along them when provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺).

In strand sequencing, the polynucleotide is translocated through the pore either with or against an applied potential. Exonucleases that act progressively or processively on double stranded polynucleotides can be used on the cis side of the pore to feed the remaining single strand through under an applied potential or the trans side under a reverse potential. Likewise, a helicase that unwinds the double stranded DNA can also be used in a similar manner. A polymerase may also be used. There are also possibilities for sequencing applications that require strand translocation against an applied potential, but the DNA must be first “caught” by the enzyme under a reverse or no potential. With the potential then switched back following binding the strand will pass cis to trans through the pore and be held in an extended conformation by the current flow. The single strand DNA exonucleases or single strand DNA dependent polymerases can act as molecular motors to pull the recently translocated single strand back through the pore in a controlled stepwise manner, trans to cis, against the applied potential.

Any helicase may be used in the method. Helicases may work in two modes with respect to the pore. First, the method is preferably carried out using a helicase such that it moves the polynucleotide through the pore with the field resulting from the applied voltage. In this mode the 5′ end of the polynucleotide is first captured in the pore, and the helicase moves the polynucleotide into the pore such that it is passed through the pore with the field until it finally translocates through to the trans side of the membrane. Alternatively, the method is preferably carried out such that a helicase moves the polynucleotide through the pore against the field resulting from the applied voltage. In this mode the 3′ end of the polynucleotide is first captured in the pore, and the helicase moves the polynucleotide through the pore such that it is pulled out of the pore against the applied field until finally ejected back to the cis side of the membrane.

The method may also be carried out in the opposite direction. The 3′ end of the polynucleotide may be first captured in the pore and the helicase may move the polynucleotide into the pore such that it is passed through the pore with the field until it finally translocates through to the trans side of the membrane.

When the helicase is not provided with the necessary components to facilitate movement or is modified to hinder or prevent its movement, it can bind to the polynucleotide and act as a brake slowing the movement of the polynucleotide when it is pulled into the pore by the applied field. In the inactive mode, it does not matter whether the polynucleotide is captured either 3′ or 5′ down, it is the applied field which pulls the polynucleotide into the pore towards the trans side with the enzyme acting as a brake. When in the inactive mode, the movement control of the polynucleotide by the helicase can be described in a number of ways including ratcheting, sliding and braking. Helicase variants which lack helicase activity can also be used in this way.

The polynucleotide may be contacted with the polynucleotide binding protein and the pore in any order. It is preferred that, when the polynucleotide is contacted with the polynucleotide binding protein, such as a helicase, and the pore, the polynucleotide firstly forms a complex with the protein. When the voltage is applied across the pore, the polynucleotide/protein complex then forms a complex with the pore and controls the movement of the polynucleotide through the pore.

Any steps in the method using a polynucleotide binding protein are typically carried out in the presence of free nucleotides or free nucleotide analogues and an enzyme cofactor that facilitates the action of the polynucleotide binding protein. The free nucleotides may be one or more of any of the individual nucleotides discussed above. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

The molecular brakes may be any compound or molecule which binds to the polynucleotide and slows the movement of the polynucleotide through the pore. The molecular brake may be any of those discussed above. The molecular brake preferably comprises a compound which binds to the polynucleotide. The compound is preferably a macrocycle. Suitable macrocycles include, but are not limited to, cyclodextrins, calixarenes, cyclic peptides, crown ethers, cucurbiturils, pillararenes, derivatives thereof or a combination thereof. The cyclodextrin or derivative thereof may be any of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J Am. Chem. Soc. 116, 6081-6088. The cyclodextrin is more preferably heptakis-6-amino-β-cyclodextrin (am₇-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

Spacers in the Target Polynucleotide

If a helicase is used in the invention, it may be stalled at one or more spacers as discussed in International Application No. PCT/GB2014/050175 (published as WO 2014/135838). Any configuration of one or more helicases and one or more spacers disclosed in the International Application may be used in this invention.

Double Stranded Polynucleotide

If a target polynucleotide is double stranded, the method preferably further comprises providing the target polynucleotide with a hairpin loop at one end of the polynucleotide. The method may comprise linking the two strands of the target polynucleotide at one end with a hairpin loop. The pore and optionally the molecular brake preferably separates the two strands of the target polynucleotide and controls the movement of the target polynucleotide through the pore one strand at a time. Linking and interrogating both strands on a double stranded construct in this way increases the efficiency and accuracy of characterisation.

Suitable hairpin loops can be designed using methods known in the art. The hairpin loop may be any length. The hairpin loop is typically 110 or fewer nucleotides, such as 100 or fewer nucleotides, 90 or fewer nucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides, 60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides, 30 or fewer nucleotides, 20 or fewer nucleotides or 10 or fewer nucleotides, in length. The hairpin loop is preferably from about 1 to 110, from 2 to 100, from 5 to 80 or from 6 to 50 nucleotides in length. Longer lengths of the hairpin loop, such as from 50 to 110 nucleotides, are preferred if the loop is involved in the differential selectability of the adaptor. Similarly, shorter lengths of the hairpin loop, such as from 1 to 5 nucleotides, are preferred if the loop is not involved in the selectable binding as discussed below.

The hairpin loop may be provided at either end of the polynucleotide, i.e. the 5′ or the 3′ end. The hairpin loop may be ligated to the polynucleotide using any method known in the art. The hairpin loop may be ligated using a ligase, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase.

The two strands of the polynucleotide may be separated using any method known in the art. For instance, they may be separated by a molecular brake, such as a polynucleotide binding protein, or using conditions which favour dehybridsation (examples of conditions which favour dehybridisation include, but are not limited to, high temperature, high pH and the addition of agents that can disrupt hydrogen bonding or base pairing, such as formamide and urea).

The hairpin loop preferably comprises a selectable binding moiety. This allows the polynucleotide to be purified or isolated. A selectable binding moiety is a moiety that can be selected on the basis of its binding properties. Hence, a selectable binding moiety is preferably a moiety that specifically binds to a surface. A selectable binding moiety specifically binds to a surface if it binds to the surface to a much greater degree than any other moiety used in the invention. In preferred embodiments, the moiety binds to a surface to which no other moiety used in the invention binds.

Suitable selective binding moieties are known in the art. Preferred selective binding moieties include, but are not limited to, biotin, a polynucleotide sequence, antibodies, antibody fragments, such as Fab and ScSv, antigens, polynucleotide binding proteins, poly histidine tails and GST tags. The most preferred selective binding moieties are biotin and a selectable polynucleotide sequence. Biotin specifically binds to a surface coated with avidins. Selectable polynucleotide sequences specifically bind (i.e. hybridise) to a surface coated with homologus sequences. Alternatively, selectable polynucleotide sequences specifically bind to a surface coated with polynucleotide binding proteins.

The hairpin loop and/or the selectable binding moiety may comprise a region that can be cut, nicked, cleaved or hydrolysed. Such a region can be designed to allow the polynucleotide to be removed from the surface to which it is bound following purification or isolation. Suitable regions are known in the art. Suitable regions include, but are not limited to, an RNA region, a region comprising desthiobiotin and streptavidin, a disulphide bond and a photocleavable region.

Leader Sequence

The target polynucleotide may be provided with a leader sequence which preferentially threads into the pore or which is capable of selectively attaching to the preceding target polynucleotide. The leader sequence facilitates the method of the invention. The leader sequence may be designed to preferentially thread into the transmembrane pore and thereby facilitate the movement of polynucleotide through the pore. The leader sequence may be designed such that it selectively attaches to the preceding target polynucleotide and facilitates the formation of the concatenated polynucleotide. The leader sequence can also be used to link the polynucleotide to the one or more anchors as discussed above.

The leader sequence typically comprises a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader preferably comprises a polynucleotide and more preferably comprises a single stranded polynucleotide. The leader sequence can comprise any of the polynucleotides discussed above. The single stranded leader sequence most preferably comprises a single strand of DNA, such as a poly dT section. The leader sequence preferably comprises the one or more spacers.

The leader sequence can be any length, but is typically 10 to 150 nucleotides in length, such as from 20 to 150 nucleotides in length. The length of the leader typically depends on the transmembrane pore used in the method.

The leader sequence in a subsequent target polynucleotide typically comprises the part which hybridizes to the part in the preceding target polynucleotide. The leader sequence preferably comprises one part of the click chemistry, such as a click reactive group.

Y Adaptors

A double stranded target polynucleotide may be provided with adaptors at one or both ends. A double stranded target polynucleotide may be provided with a Y adaptor at both ends.

A double stranded target polynucleotide may be provided with a Y adaptor at one end and a hairpin loop at the other end. A method of characterising a polynucleotide may comprise attaching a Y adaptor to one end of a double stranded target polynucleotide and attaching a hairpin loop at the other end. The Y adaptor and/or the hairpin adaptor are typically polynucleotide adaptors. They may be formed from any of the polynucleotides discussed above.

The Y adaptor typically comprises (a) a double stranded region and (b) a single stranded region or a region that is not complementary at the other end. The Y adaptor may be described as having an overhang if it comprises a single stranded region. The presence of a non-complementary region in the Y adaptor gives the adaptor its Y shape since the two strands typically do not hybridise to each other unlike the double stranded portion. In other words, the Y adaptor comprises two polynucleotide strands, e.g. DNA strands. A portion extending to the 3′ end of the first strand is complementary to a portion extending to the 5′ end of the other strand. The complementary portions hybridise to each other and form a duplex, or double stranded region of the Y adaptor. The remaining portions of the first and second strands are not complementary and do not hybridise to one another.

In one embodiment, the Y adaptor comprises one or more attachment sites that can be used to selectively attach a first (preceding) target polynucleotide to a second (subsequent) target polynucleotide as described herein.

In one embodiment, a first attachment site is present in the double stranded region of the Y adaptor. When the Y adaptor is ligated to the end of a target polynucleotide, the attachment site in the double stranded region is “hidden” until the target polynucleotide interacts with a pore. This “hidden” attachment site corresponds to the part of the first (preceding) target polynucleotide that can be selectively attached to a part of the second (subsequent) target polynucleotide.

In one embodiment, a second attachment site is present in the single stranded region of the Y adaptor. When the Y adaptor is ligated to the end of a second target polynucleotide, the attachment site in the single stranded region is available to bind to a first attachment site in a first target polynucleotide when the first target polynucleotide interacts with a pore to reveal the first attachment site. This “exposed” attachment site corresponds to the part of the second (subsequent) target polynucleotide that can be selectively attached to a part of the first (preceding) target polynucleotide.

A Y adaptor may comprise a first attachment site and a second attachment site as described above. In one embodiment, the first and second attachment sites present in a Y adaptor are not complementary to each other. A population of different Y adaptors, wherein complementary attachment sites are present in different Y adaptors, may then be used to perform a method as described herein. In an example of this embodiment, the first (hidden) attachment site and the second (exposed) attachment site in a first Y adaptor may both have the same sequence, which sequence is complementary to the sequence of a first (hidden) attachment site and a second (exposed) attachment site in a second Y adaptor. The first and second Y adaptors may then be used together, wherein a first (preceding) target polynucleotide comprising the first Y adaptor is concatenated to a second (subsequent) target polynucleotide that comprises the second Y adaptor. In another embodiment, the first and second attachment sites present in a Y adaptor are complementary to each other. In this embodiment, a single type of Y adaptor may be used.

The invention provides a Y adaptor comprising a first polynucleotide strand and a second polynucleotide strand, wherein: (i) a portion extending to the 3′ end of the first polynucleotide strand is complementary to a portion extending to the 5′ end of the second polynucleotide strand and the complementary portions form a duplex; (ii) a portion extending to the 5′ end of the first polynucleotide strand and a portion extending to the 3′ end of the second polynucleotide strand do not hybridise to one another; and (iii) the portion extending to the 5′ end of the second polynucleotide strand comprises a sequence that is capable, when the duplex is unwound, of hybridising to a sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand.

The sequence in the portion extending to the 5′ end of the second polynucleotide strand and the sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand that are capable of hybridising to each other preferably have a length of from 6 to 50 base pairs, such as from 7 to 40, 8 to 30, 9 to 20 or 10 to 15 base pairs.

The invention also provides a Y adaptor comprising a first polynucleotide strand and a second polynucleotide strand, wherein: (i) a portion extending to the 3′ end of the first polynucleotide strand is complementary to a portion extending to the 5′ end of the second polynucleotide strand and the complementary portions form a duplex; (ii) a portion extending to the 5′ end of the first polynucleotide strand and a portion extending to the 3′ end of the second polynucleotide strand do not hybridise to one another; and (iii) the portion extending to the 5′ end of the second polynucleotide strand comprises a sequence that is identical to a sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand.

The sequence in the portion extending to the 5′ end of the second polynucleotide strand that is identical to the sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand preferably has a length of from 6 to 50 base pairs, such as from 7 to 40, 8 to 30, 9 to 20 or 10 to 15 base pairs.

The duplex region in the Y adaptor may have a length of from about 6 to 200 base pairs, such as from 10 to 150, 20 to 175, 25 to 150, 50 to 125 or 75 to 100 base pairs. The duplex region may comprise a blocker sequence to prevent movement of a helicase along the duplex strand. In one embodiment, the blocker comprises iSp18. Suitable blocker sequences are described in the Examples.

In the Y adaptor, the portion extending to the 5′ end of the first polynucleotide strand is preferably from 10 to 100 base pairs in length, such as from 10 to 75, 20 to 65 or 25 to 50 base pairs in length. The portion extending to the 5′ end of the first polynucleotide strand comprises an exposed attachment site. The portion extending to the 5′ end of the first polynucleotide strand preferably comprises a polymer leader sequence. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader sequence preferably comprises one or more spacers, such as iSpC3. Examples of suitable leader sequences are described in the Examples.

The leader sequence can be any length, but is typically 10 to 150 nucleotides in length, such as from 20 to 150, 25 to 100 or 30 to 50 nucleotides in length. The length of the leader typically depends on the transmembrane pore used in the method. In one embodiment the combined length of the leader sequence and the exposed attachment site, which may comprise all or part of the leader sequence is from 25 to 40, such as 30 base pairs.

In one embodiment, the 5′ end of the first polynucleotide strand of the adaptor comprises comprises a first part of the click chemistry, such as a click reactive group.

In one embodiment, the 3′ end of the second polynucleotide strand of the adaptor comprises a second part of the click chemistry, such as a click reactive group. The first part of the click chemistry is a part that reacts with the second part of the click chemistry. Typically, in this embodiment, the portion extending to the 5′ end of the second polynucleotide strand comprises a sequence that is capable, when the duplex is unwound, of hybridising to a sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand.

In another embodiment, the 3′ end of the second polynucleotide strand of the adaptor comprises a first part of the click chemistry, such as a click reactive group. In this embodiment the adaptor is designed for use together with a second Y adaptor which comprises a second part of the click chemistry, such as a click reactive group at the 5′ end of its first polynucleotide strand and a second part of the click chemistry, such as a click reactive group at the 3′ end of its second polynucleotide strand. Typically, in this embodiment, the portion extending to the 5′ end of the second polynucleotide strand comprises a sequence that is identical to a sequence comprised in the a portion extending to the 5′ end of the first polynucleotide strand.

In one embodiment, the 5′ end of the first polynucleotide strand of the adaptor comprises a phosphatase. The phosphatase may facilitate ligation of the 5′ end of the first polynucleotide strand to the 3′ end of another polynucleotide strand (typically the 3′ end of the second strand of a second adaptor after exposure of the hidden attachment site in the second adaptor).

The Y adaptor may comprise one or more anchors. Anchors are discussed in more detail above. In one embodiment, the Y adaptor preferably comprises an anchor attached to a third polynucleotide strand which comprises a 5′ region that hybridises to the first polynucleotide strand. The part of the first polynucleotide strand to which the third polynucleotide strand binds is typically between the region of the first polynucleotide strand that hybridises to the second polynucleotide strand and the leader sequence. The anchor is preferably attached at or close to the 3′ end of the third polynucleotide strand. A fourth polynucleotide strand may be hybridized to a region of the third polynucleotide strand that is not hybridised to the first polynucleotide strand. The anchor is preferably cholesterol.

One part of the non-complementary region in the second strand of each Y adaptor preferably forms a loop structure. This facilitates the method of invention as discussed above. In particular it facilitates the action of ligases that join double stranded polynucleotides. In this embodiment, the two sides of the loop typically hybridise to one another. The nucleotide base at the 3′ end of the second polynucleotide strand is preferably at the end of the loop. The nucleotide base at the 3′ end of the second polynucleotide strand is typically hybridised to another part of the second polynucleotide strand. The first (hidden) attachment site is preferably adjacent to, but not part of the loop structure. Hybridisation of the first (hidden) attachment site to the second (exposed) attachment site in a different Y adaptor elongates the loop. The Y adaptors, which are at the ends of target polynucleotides, can then be joined, for example by ligation (using a ligase) or by click chemistry. This embodiment results in the two adaptors (and hence the preceding and subsequent target polynucleotides) being attached by a loop which may dehybridise and move through the pore.

The loop in the second polynucleotide strand may be any length. The loop in the second polynucleotide strand preferably comprises 10 to 50 nucleotides, such as from 15 to 40, or 20 to 25, for example 23 nucleotides.

The Y adaptor preferably comprises a leader sequence which preferentially threads into the pore. Leader sequences are discussed above. In one embodiment a second (exposed) attachment site is present in the same strand of the Y adaptor as a leader sequence. Typically, the leader sequence is at the 5′ end of the Y adaptor and the second attachment site is present between the leader sequence and the double stranded region. The exposed attachment site may form part of the leader sequence.

The Y adaptor may further comprise one or more polynucleotide binding proteins. In one embodiment a polynucleotide binding protein is attached to the first polynucleotide strand of the Y adaptor. The polynucleotide binding protein is preferably located 3′ of the leader sequence and 5′ to the blocker sequence. The polynucleotide binding protein is preferably a helicase. More than one, such as two or more polynucleotide binding proteins, preferably two or more helicases, may be comprised in the adaptor. The two or more polynucleotide binding proteins are preferably different from one another, for example are two different helicases. Where two polynucleotide binding proteins are present, the two proteins preferably process the bound polynucleotide at different rates once the effect of the blocker sequence is removed. For example, a first polynucleotide binding protein, preferably a first helicase, may be attached to the first polynucleotide strand of the adaptor 3′ to a second polynucleotide binding protein, preferably a second helicase. In this example, the first polynucleotide binding protein may separate the two strands of a double stranded target polynucleotide and the second polynucleotide binding protein may act to control movement of the target polynucleotide through a nanopore. In this embodiment, the first polynucleotide binding protein may be referred to as a “release protein” and the second polynucleotide binding protein as a “motor protein”. The release protein preferably moves along the polynucleotide faster than the motor protein.

The Y adaptor preferably comprises a selectable binding moiety as discussed above. The Y adaptor and/or the selectable binding moiety may comprise a region that can be cut, nicked, cleaved or hydrolysed as discussed above.

The Y adaptor and/or the hairpin loop may be ligated to the polynucleotide using any method known in the art. One or both of the adaptors may be ligated using a ligase, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase. Alternatively, the adaptors may be added to the polynucleotide using the methods of the invention discussed below.

In a preferred embodiment, the method comprises modifying the double stranded polynucleotide so that it comprises adaptors at its ends. For example, in one embodiment, the method comprises modifying the double stranded polynucleotide so that it comprises the Y adaptor at one end and the hairpin loop at the other end. In another embodiment, the method comprises modifying the double stranded polynucleotide so that it comprises a Y adaptor at each end. Any manner of modification can be used. The method preferably comprises modifying the double stranded polynucleotide in accordance with the invention. This is discussed in more detail below. The methods of modification and characterisation may be combined in any way.

Adding Hairpin Loops and Leader Sequences

The double stranded polynucleotide may be provided with Y adaptors and hairpin loops by contacting the polynucleotide with a MuA transposase and a population of double stranded MuA substrates, wherein a proportion of the substrates in the population are Y adaptors comprising the leader sequence and wherein a proportion of the substrates in the population are hairpin loops. The transposase fragments the double stranded polynucleotide and ligates MuA substrates to one or both ends of the fragments. This produces a plurality of modified double stranded polynucleotides comprising the leader sequence at one end and the hairpin loop at the other. The modified double stranded polynucleotides may then be investigated using the method of the invention.

These MuA based methods are disclosed in International Application No. PCT/GB2014/052505 published as (WO 2015/022544). They are also discussed in detail in International Application No PCT/GB2015/050991.

The double stranded polynucleotide may be provided with Y adaptors by contacting the polynucleotide with a MuA transposase and a population of double stranded MuA substrates, wherein the substrates are Y adaptors comprising a leader sequence and attachment sites. The transposase fragments the double stranded polynucleotide and ligates MuA substrates to one or both ends of the fragments. This produces a plurality of modified double stranded target polynucleotides comprising single stranded overhangs at both ends. The modified double stranded polynucleotides may then be investigated using the method of the invention.

Modified Polynucleotide Analytes

Before characterisation, the polynucleotide may be modified by contacting the polynucleotide with a polymerase and a population of free nucleotides under conditions in which the polymerase forms a modified polynucleotide using the polynucleotide as a template, wherein the polymerase replaces one or more of the nucleotide species in the polynucleotide with a different nucleotide species when forming the modified polynucleotide analyte. The modified polynucleotide may then be characterised in accordance with the invention. This type of modification is described in PCT Application No. PCT/GB2015/050483. Any of the polymerases discussed above may be used. The polymerase is preferably Klenow or 90 North.

Population of Y Adaptors

The invention also provides a population of two or more polynucleotide Y adaptors, wherein each adaptor comprises first and second parts (first and second attachment sites) which are capable of hybridising together and wherein each first part (first attachment site) is initially protected from hybridisaton to the second part (second attachment site). The population may comprise any number of adaptors, such as the numbers discussed above for target polynucleotide.

Also provided is a population of polynucleotide adaptors comprising a first polynucleotide adaptor and a second polynucleotide adaptor, wherein the first polynucleotide adaptor and the second polynucleotide adaptor each comprise a first polynucleotide strand and a second polynucleotide strand, wherein: (a)(i) a portion extending to the 3′ end of the first polynucleotide strand is complementary to a portion extending to the 5′ end of the second polynucleotide strand and the complementary portions form a duplex; (ii) a portion extending to the 5′ end of the first polynucleotide strand and a portion extending to the 3′ end of the second polynucleotide strand do not hybridise to one another; and wherein (b) the portion extending to the 5′ end of the second polynucleotide strand of the first polynucleotide adaptor comprises a sequence that is capable, when the duplex is unwound, of hybridising to a sequence comprised in the portion extending to the 5′ end of the first polynucleotide strand of the second polynucleotide adaptor. These polynucleotide adaptors are referred to a “Y adaptors” because of their shape.

The Y adaptors in the population may have any of the features described in the section “Y adaptors” above. Any of the embodiments discussed above with reference to the methods of the invention equally apply to the population of the invention.

As discussed above, each Y adaptor typically comprises (a) a double stranded region and (b) a single stranded region or a region that is not complementary at the other end. The presence of the non-complementary region in the Y adaptor gives the adaptor its Y shape since the two part/strands typically do not hybridise to each other unlike the double stranded portion.

In other words, the Y adaptor comprises two polynucleotide strands, e.g. DNA strands. A portion extending to the 3′ end of the first strand is complementary to a portion extending to the 5′ end of the other strand. The complementary portions hybridise to each other and form a duplex, or double stranded region of the Y adaptor. The remaining portions of the first and second strands are not complementary and do not hybridise to one another.

In one embodiment, the Y adaptor comprises one or more attachment sites that can be used to selectively attach a first (preceding) target polynucleotide to a second (subsequent) target polynucleotide as described herein.

In one embodiment, a first attachment site is present in the double stranded region of the Y adaptor. When the Y adaptor is ligated to the end of a target polynucleotide, the attachment site in the double stranded region is “hidden” until the target polynucleotide interacts with a pore. This “hidden” attachment site corresponds to the part of the first (preceding) target polynucleotide that can be selectively attached to a part of the second (subsequent) target polynucleotide.

In one embodiment, a second attachment site is present in the single stranded region of the Y adaptor. When the Y adaptor is ligated to the end of a second target polynucleotide, the attachment site in the single stranded region is available to bind to a first attachment site in a first target polynucleotide when the first target polynucleotide interacts with a pore to reveal the first attachment site. This “exposed” attachment site corresponds to the part of the second (subsequent) target polynucleotide that can be selectively attached to a part of the first (preceding) target polynucleotide.

A Y adaptor may comprise a first attachment site and a second attachment site as described above. In one embodiment, the first and second attachment sites present in a Y adaptor are not complementary to each other. A population of different Y adaptors, wherein complementary attachment sites are present in different Y adaptors, may then be used to perform a method as described herein. In another embodiment, the first and second attachment sites present in a Y adaptor are complementary to each other. In this embodiment, a single type of Y adaptor may be used.

The Y adaptor may comprise one or more anchors. Anchors are discussed in more detail above. In one embodiment, the Y adaptor preferably comprises an anchor attached to the portion extending to the 3′ end of the second polynucleotide strand.

The first part (first attachment site) is preferably initially protected by hybridisaton to the opposite strand in the double stranded region of the Y adaptor.

One half of the non-complementary region in each Y adaptor preferably forms a loop structure. This facilitates the method of invention as discussed above. The first part (is preferably adjacent to, but not part of the loop structure. Hybridisation of the first part to the second part in a different Y adaptor elongates the loop. This embodiment results in the two adaptors (and hence the preceding and subsequent target polynucleotides) being attached by a loop which may dehybridise and move through the pore.

Each Y adaptor preferably further comprises a leader sequence comprising the second part. Suitable leader sequences are discussed above. The second part is preferably an overhang formed by a bridging polynucleotide hybridised to the free end of the leader sequence. The bridging polynucleotide may be any length and formed from any of the types of polynucleotide discussed above. In one embodiment the second (exposed) attachment site is present in the same strand of the Y adaptor as a leader sequence. Typically, the leader sequence is at the 5′ end of the Y adaptor and the second attachment site is present between the leader sequence and the double stranded region.

In a preferred embodiment, only one of the adaptors in the population comprises a molecular brake. Attachment of this adaptor to the first target polynucleotide means that the molecular brake will control the movement of the concatenated polynucleotide, i.e. all of the target polynucleotides, through the pore.

The first part preferably comprises a click reactive group and the second part comprises the complementary click reactive group. The reactive groups may be any of those discussed above.

Kits

The present invention also provides a kit for characterising two or more double stranded target polynucleotides. In one embodiment, the kit comprises a population of Y adaptors of the invention. In a further embodiment, the kit comprises a Y adaptor of the invention. In another embodiment, the kit comprises a population of Y adaptors of the invention and a population of hairpin loops. Such loops are discussed above.

The kit may further comprise a microparticle for delivering the target polynucleotides to a transmembrane pore in a membrane. The kit may further comprise one or more anchors which are capable of coupling the polynucleotides to a membrane. The microparticle and the one or more anchors may be any of those discussed above with reference to the method of the invention. The microparticle is preferably part of the kit for extracting and/or purifying the polynucleotide.

Any of the embodiments discussed above with reference to the method of the invention equally apply to the kits. The kit may further comprise the components of a membrane, such as the components of an amphiphilic layer or a triblock copolymer membrane. The kit may further comprise a transmembrane protein pore.

The kit of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. The kit may comprise a magnet or an electromagnet. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and/or express polynucleotides, a membrane as defined above or voltage or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding for which organism the method may be used.

The following Examples illustrate the invention.

EXAMPLES Example 1

This example describes a method of characterising a concatenated polynucleotide where the method of attachment used to join the polynucleotides together is by ligation.

Materials and Methods

Ligation Prep

A leader strand (ID 1 below), a bottom strand (ID NO: 2 below), containing a 3′ hairpin, and a blocker strand (ID No: 3 below) were annealed at 5 μM, 6 μM and 6 μM respectively in 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at 2° C. per minute. The hybridised DNA was known as adapter 1.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where (ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on ice before 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mM potassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according to the manufacturer's instructions. The recovered protein was quantified using the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the same buffer. 27 ul of buffer exchanged protein was mixed with 3 ul of adapter 1 in a DNA low bind eppendorf and left to incubate for 10 mins at 35° C. 0.37 ul of 8.1 mM TMAD was then added and the sample was left to incubate for 60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mM MgCl2, 2 mM rATP was then added and left for a further 20 mins at room temperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were then added and the sample incubated for 5 mins at room temperature on a rotator. The beads were pelleted on a magnetic rack and the supernatant removed. While still on the magnetic rack the beads were washed with 500 ul of 50 mM Tris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathe the pellet on the rack. The wash buffer was removed and the pellet pulsed briefly in a centrifuge before returning to the magnetic rack to remove the last remnants of solution. The pellet was then resuspended in 30 ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperature before being placed on a magnetic rack to recover the purified adapter which was known as preloaded Y-adapter 1.

500 ng of end-repaired and dA-tailed E. coli genomic DNA was ligated for 10 mins at room temperature in 50 ul with 5 ul of 200 nM preloaded Y-adapter 1 from above and 1 ul of 1 μM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) was added and the sample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to a magnetic rack and the supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. The beads were then added to the ligation mix and incubated on a rotator for 10 mins at room temperature. The bead bound library was then added to a magnetic rack and the supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5 ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nM of tether (ID NO: 5). The sample was incubated for 10 mins at 37° C. before the beads were pelleted and the library containing the supernatant was removed.

Electrical measurements were acquired from single MspA nanopores inserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0). After achieving a single pore inserted in the block co-polymer, then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) was flowed through the system to remove any excess MspA nanopores. 500 ul of 25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mM rATP, with 10 mins between each wash. 12 ul of the recovered bead purified library was added to 150 ul of 50 mM potassium phosphate buffer pH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP, 124 ul of nuclease free water and 6 ul of T3 DNA ligase (NEB). 150 ul of this sequencing mix was then added to the nanopore system. The experiment was run at −140 mV and helicase-controlled DNA movement monitored.

ID NO: 1 (SEQ ID NO: 28) /5Phos/GCGGTTGTT/iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3/ (SEQ ID NO: 29) /iSp18//iSp18//iSp18//iSp18/ ID NO: 2 (SEQ ID NO: 30) /5Phos/ (SEQ ID NO: 31) /iSp18//iSp18//iSp18/ (SEQ ID NO: 32) /iSp18//iSp18//iSp18/ ID NO: 3 (SEQ ID NO: 33) /5BNA-GMBNA-GMBNA-T/ABNA-T//iBNA-A/ ID NO: 4 (SEQ ID NO: 34) /5Phos/ (SEQ ID NO: 35) /iSp18//iSp18//iSp18/ (SEQ ID NO: 36) /iSp18//iSp18//iSp18/ ID NO: 5 (SEQ ID NO: 37) /5CholTEG/  ID NO: 6 (SEQ ID NO: 38) /5desthiobiotinTEG/TT/iSp18//iSp18//iSp18//iSp18// iSp18//iSp18/ /iSp18//iSp18//iSp18//iSp18//iSp18//iSp18/TT/ 3CholTEG Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to control the movement of the concatenated polynucleotide through the MspA nanopore. FIGS. 2-6 each show a current trace (top trace on each slide) of a concatenated polynucleotide (a first polynucleotide attached to one subsequent polynucleotide) as it translocated through the nanopore. The lower trace of FIGS. 2-6 show zoomed in regions 1-5 of the upper trace. The lower traces show translocation of spacer groups (found in the leader and hairpin regions of the first and subsequent polynucleotide and marked with a *) through the nanopore. The spacer groups allowed more current to flow through the nanopore as they translocated through it. The example trace shows that the first polynucleotide was successfully ligated to the subsequent polynucleotide.

Example 2

This example describes a method of characterising a concatenated polynucleotide where the method of attachment used to join the polynucleotides together is by click chemistry (see FIG. 7 for a cartoon representation of attachment using click chemistry).

Materials and Methods

Click Ligation Prep:

A leader strand (ID NO: 7) and a blocker strand (ID NO: 8), containing a tether hybridisation site, were annealed at 5.5 μM and 6 μM respectively in 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at 2° C. per minute. 10 μM of a bottom strand (ID NO: 9), containing a 3′ hairpin, was heated to 95° C. for 1 min before being snap cooled on ice in 50 mM HEPES pH 8, 100 mM potassium acetate. The two samples were equilibrated to 50° C. before being mixed 1:1 and left at 40° C. for 1 min before snap cooling on ice. The hybridised DNA was known as adapter 2.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where (ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on ice before 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mM potassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according to the manufacturer's instructions. The recovered protein was quantified using the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the same buffer.

27 ul of buffer exchanged protein was mixed with 3 ul of adapter 2 in a DNA low bind eppendorf and left to incubate for 10 mins at 35° C. 0.37 ul of 8.1 mM TMAD was then added and the sample was left to incubate for 60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mM MgCl2, 2 mM rATP was then added and left for a further 20 mins at room temperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were added and the sample incubated for 5 mins at room temperature on a rotator. The beads were pelleted on a magnetic rack and the supernatant removed. While still on the magnetic rack the beads were washed with 500 ul of 50 mM Tris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathe the pellet on the rack. The wash buffer was removed and the pellet pulsed briefly in a centrifuge before returning to the magnetic rack to remove the last remnants of solution. The pellet was resuspended in 30 ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperature before being placed on a magnetic rack to recover the purified adapter which was known as preloaded Y-adapter 2.

500 ng of end-repaired and dA-tailed E. coli genomic DNA was ligated for 10 mins at room temperature in 50 ul with 5 ul of 200 nM preloaded Y-adapter 2 from above and 1 ul of 1 uM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) was added and the sample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. The beads were then added to the ligation mix and incubated on a rotator for 10 mins at room temperature. The bead bound library was then added to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5 ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nM of tether (ID NO: 5). The sample was incubated for 10 mins at 37° C. before the beads pelleted and the library containing supernatant was removed.

Electrical measurements were acquired from single MspA nanopores inserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0). After achieving a single pore inserted in the block co-polymer, then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) was flowed through the system to remove any excess MspA nanopores. 500 ul of 25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mM rATP, with 10 mins between each wash. 12 ul of the recovered bead purified library was added to 150 ul of 50 mM potassium phosphate buffer pH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP and 130 ul of nuclease free water. 150 ul of this sequencing mix was then added to the nanopore system. The experiment was run at −140 mV and helicase-controlled DNA movement monitored.

ID NO: 7 (SEQ ID NO: 39) /Azide/ (SEQ ID NO: 28) /iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3/  (SEQ ID NO: 29) /iSp18//iSp18//iSp18//iSp18/  ID NO: 8 SEQ ID NO: 40 ID NO: 9 (SEQ ID NO: 41) /5Phos/ (SEQ ID NO: 32) /iSp18//iSp18//iSp18//iSp18/ /DBCO/ Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to control the movement of the concatenated polynucleotide through the MspA nanopore. FIG. 8 shows a current trace (top trace) of a concatenated polynucleotide (a first polynucleotide attached to five subsequent polynucleotides) as it translocated through the nanopore. The lower traces of FIG. 8 show zoomed in regions a*-k* of the upper trace. The lower traces show translocation of spacer groups (found in the leader and hairpin regions of the first and subsequent polynucleotides and marked with an a*-k*) through the nanopore. The spacer groups allowed more current to flow through the nanopore as they translocated through it. The example trace shows that the first polynucleotide was successfully attached to five subsequent polynucleotides using click chemistry.

Example 3

This example describes a method of characterising a concatenated polynucleotide where the method of attachment used to join the polynucleotides together is by click chemistry. In this example one group of adapters has a pre-bound enzyme which was used to produce a seed library and the second group has no enzyme bound which was used to produce a sequencing library.

Seed Library Prep:

A seed library Y-adapter was produced using a leader strand (ID NO: 10), a bottom strand (ID NO: 9), containing a 3′ hairpin, and a blocker strand (ID NO: 8), containing a tether hybridisation site, which were annealed at 5.5 μM, 6 μM and 5 μM respectively in 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at 2° C. per minute. The hybridised DNA was known as adapter 2.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where (ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on ice before 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mM potassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according to the manufacturer's instructions. The recovered protein was quantified using the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the same buffer.

27 ul of buffer exchanged protein was mixed with 3 ul of adapter 2 in a DNA low bind eppendorf and left to incubate for 10 mins at 35° C. 0.37 ul of 8.1 mM TMAD was then added and the sample was left to incubate for 60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mM MgCl2, 2 mM rATP was then added and left for a further 20 mins at room temperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were added and the sample incubated for 5 mins at room temperature on a rotator. The beads were pelleted on a magnetic rack and the supernatant removed. While still on the magnetic rack the beads were washed with 500 ul of 50 mM Tris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathe the pellet on the rack. The wash buffer was removed and the pellet pulsed briefly in a centrifuge before returning to the magnetic rack to remove the last remnants of solution. The pellet was resuspended in 30 ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperature before being placed on a magnetic rack to recover the purified adapter which was known as preloaded Seed Y-adapter 2.

A sequencing library Y-adapter was produced by hybridising a leader strand (ID NO: 7) and a blocker strand (ID NO: 11), containing a tether hybridisation site and a polyA 5′ extension, at 250 nM and 300 nM respectively in 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at 2° C. per minute. 400 nM of a bottom strand (ID NO: 9), containing a 3′ hairpin, was heated to 95° C. for 1 min before being snap cooled on ice in 50 mM HEPES pH 8, 100 mM potassium acetate. The two samples were equilibrated to 50° C. before being mixed 1:1 and left at 40° C. for 1 min before snap cooling on ice. This sample was known as enzyme free sequencing library Y-adapter 3.

A seed library was set up by ligating 500 ng of end-repaired and dA-tailed E. coli genomic DNA for 10 mins at room temperature in 50 ul with 5 ul of 200 nM preloaded seed Y-adapter 2 from above and 1 ul of 1 μM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) was added and the sample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. The beads were then added to the ligation mix and incubated on a rotator for 10 mins at room temperature. The bead bound library was then added to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5 ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nM of tether (ID NO: 5). The sample was incubated for 10 mins at 37° C. before the beads were pelleted and the library containing supernatant was removed.

A sequencing library was set up by ligating 500 ng of end-repaired and dA-tailed E. coli genomic DNA for 10 mins at room temperature in 50 ul with 5 ul of 200 nM enzyme free sequencing library Y-adapter 3 from above and 1 ul of 1 μM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) was added and the sample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. The beads were then added to the ligation mix and incubated on a rotator for 10 mins at room temperature. The bead bound library was then added to a magnetic rack and supernatant was removed. The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5 ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nM of tether (ID NO: 5). The sample was incubated for 10 mins at 37° C. before the beads were pelleted and the library containing supernatant was removed.

Electrical measurements were acquired from single MspA nanopores inserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0). After achieving a single pore inserted in the block co-polymer, then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) was flowed through the system to remove any excess MspA nanopores. 500 ul of 25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mM rATP, with 10 mins between each wash. 6 ul of the recovered bead purified seed library was added to 150 ul of 50 mM potassium phosphate buffer pH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP and 130 ul of nuclease free water. Following thorough mixing by inversion, 6 ul of the sequencing library was added and the sample again mixed thoroughly by inversion. 150 ul of this sequencing mix was then added to the nanopore system. The experiment was run at −140 mV and helicase-controlled DNA movement monitored for 6 hours.

ID NO: 10 (SEQ ID NO: 28) /5SpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3/ (SEQ ID NO: 29) /iSp18//iSp18//iSp18//iSp18/  ID NO 11 (SEQ ID NO: 42) Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to control the movement of the concatenated polynucleotide through the MspA nanopore. Similar results were obtained as were observed for example 2.

Example 4

This example describes a method of characterising and concatenating only template strands of target polynucleotides, where the method of attachment used to join the polynucleotides together is by click chemistry.

A concatenation adapter complex that contains a motor protein and a release protein is prepared. This adapter is then ligated to both ends of a target polynucleotide. Both proteins are stalled on the ligated adapter complex until the adaptor ligated to a target polynucleotide is captured by the pore. Once a first polynucleotide has been captured, the blocking chemistry used to stall the proteins is overcome by both proteins. The motor protein then controls the interaction of the first polynucleotide with the pore, as described previously, and the release protein, which can translocate more quickly than the motor protein, separates the strands to expose a sequence (3′ hybridisation site) in the 3′ end of the adaptor linked to the end of the target polynucleotide that is complementary to a 5′ nucleic acid sequence (5′ hybridisation site) of the leader strand of an adapter complex that is ligated to a second target polynucleotide. With the 3′ hybridisation site revealed, the 5′ hybridisation site in the second target polynucleotide can then hybridise to the revealed 3′ hybridisation site and covalent coupling of the 3′ end of the first polynucleotide to the 5′ of a the second polynucleotide can occur (FIG. 10). This process then repeats for further concatenation of target polynucleotides. 

The invention claimed is:
 1. A method of characterising two or more target polynucleotides, comprising: (a) contacting a first target polynucleotide with a transmembrane pore in a membrane such that the first target polynucleotide moves through the pore; (b) sequentially attaching to the first target polynucleotide one or more subsequent target polynucleotides to provide a concatenated polynucleotide within which the target polynucleotides move through the pore in attachment order, wherein a subsequent target polynucleotide is selectively attached to the preceding target polynucleotide in the attachment order as the preceding target polynucleotide moves through the pore; and (c) taking one or more measurements which are indicative of one or more characteristics of the concatenated polynucleotide as it moves with respect to the pore.
 2. The method according to claim 1, wherein 2 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1,000,000 or more or 5,000,000 or more subsequent target polynucleotides are attached to the first target polynucleotide.
 3. The method according to claim 1, wherein a part of the preceding target polynucleotide is initially protected from attachment to the subsequent target polynucleotide and is revealed for attachment as the preceding target polynucleotide moves through the pore.
 4. The method according to claim 1, wherein a part of the subsequent target polynucleotide selectively hybridises to a part of the preceding polynucleotide and further wherein the part of the preceding target polynucleotide is initially protected from hybridisation to the part of the subsequent target polynucleotide and is revealed for hybridisation as the preceding target polynucleotide moves through the pore.
 5. The method according to claim 1, wherein a part of the subsequent target polynucleotide selectively hybridises to a part of the preceding polynucleotide and the preceding target polynucleotide is double stranded, wherein the part of the preceding target polynucleotide is in one strand and is hybridised to the other strand and wherein the part is revealed for hybridisation as the two strands separate as the preceding target polynucleotide moves through the pore.
 6. The method according to claim 1, wherein the preceding target polynucleotide is double stranded and wherein both strands of the double stranded preceding target polynucleotide are linked at one end by a hairpin loop, optionally wherein the other end of the preceding target polynucleotide comprises a leader sequence which preferentially threads into the pore.
 7. The method according to claim 1, wherein the subsequent target polynucleotide is double stranded and the two strands are linked at one end by a hairpin loop.
 8. The method according to claim 1, wherein the one or more subsequent target polynucleotides are selectively attached to the first target polynucleotide by (i) covalent attachment; (ii) a ligase; (iii) a topoisomerase; or (iv) click chemistry.
 9. The method according to claim 1, wherein the movement of the concatenated polynucleotide through the pore is controlled by a molecular brake.
 10. The method according to claim 1, wherein the transmembrane pore is a protein pore or a solid state pore.
 11. A method of sequentially passing polynucleotides through a transmembrane pore, the method comprising contacting a first target polynucleotide with the pore under conditions in which the first target polynucleotide enters the pore, wherein, following its entry, an attachment site is revealed on the first target polynucleotide, such that the first target polynucleotide attaches to a second target polynucleotide at the attachment site, thereby guiding sequential entry of the second target polynucleotide into the pore following passage of the first target polynucleotide through the pore. 